- Email: [email protected]
Colorectal cancer-associated nuclear antigen
Biochimica et Biophysica Acta 1501 (2000) 162^170 www.elsevier.com/locate/bba
Colorectal cancer-associated nuclear antigen Piotr Szymczyk a , JarosIaw Jakubik b , Wanda M. Krajewska a , Danuta Dus¨ c , Jan Berner b , Zo¢a M. Kilian¨ska a; * a
Department of Cytobiochemistry, University of 6o¨dz¨, ul. Banacha 12/16, 90-237 6o¨dz¨, Poland Department of Surgical Oncology, Medical University of 6o¨dz¨, ul. Paderewskiego 4, 93-509 6o¨dz¨, Poland Institute of Immunology and Experimental Therapy, Polish Academy of Science, ul. Weigla 12, 53-114 WrocIaw, Poland b
c
Received 28 April 1999; received in revised form 15 February 2000; accepted 29 February 2000
Abstract By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with Mr of 35^40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Colorectal cancer; Colon tumor cell line; Tumor marker; Nuclear matrix; Chromatin
1. Introduction Colorectal cancer is a common disease in the industrialized Western Europe countries and the United States [1,2]. This cancer is the second leading cause of malignant deaths in Poland [3]. The pathogenesis of colorectal cancer is of particular interest for the complex and only partially understood interaction between environmental factors and genetic background. The current model for colorectal carcinogenesis postulates a multistage progression involving an accumulation of gene mutations (APC, DCC, K-ras, p53, DNA mismatch repair genes), alterations
* Corresponding author. Fax: +48-42-635-44-84.
in gene expression (c-myc, TGFL receptor), and chromosome losses, during which regulation of cell growth is disrupted [4^8]. Recently published data indicate that mechanisms of colorectal tumorigenesis may di¡er according to tumor location [9^11]. It is an accepted opinion that cancers in the proximal colon are associated with intrinsic factors such as bile acids and sex hormones, whereas distal cancers are more closely related to environmental factors such as diet, cigarette smoking, and alcohol consumption [8]. A cellular hallmark of the transformed phenotype is an abnormal nuclear shape [12,13]. Nuclear structural alterations are commonly used as pathological markers in transformation in many types of cancer. Nuclear shape is thought to re£ect the internal nu-
0925-4439 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 0 ) 0 0 0 1 7 - X
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
clear structure and processes, and is determined mainly by the nuclear matrix (NM). This structure organizes DNA into structurally and functionally independent domains [14,15]. Since this structure plays an essential role in supporting most of nuclear functions, i.e. replication, transcription, RNA metabolism and transport, it has been postulated that during carcinogenesis its components would be altered [16,17]. The NM is the nonchromatin structural protein and ribonucleoprotein network that is resistant to nucleases, detergents and high-ionic strength bu¡ers [14,15,18,19]. The resulting structures which retain the overall shape of nuclei, are mainly composed of non-histone proteins and variable amounts of RNA and DNA [18^24]. Ultrastructural investigations have revealed that the NM can be divided into two compartments, i.e. internal matrix (internal network with residual nucleoli described as: NMi ) which contains residual nucleoli, and peripheral matrix (residual lamina-pore complex: NMp ) composed of residual lamina^pore complex [19,23,24]. It has shown that morphological and functional properties of the NM vary depending on the manner employed for its isolation [19,22^24]. Several studies have shown that an incubation of isolated nuclei near physiological temperature of 37³C stabilizes the NMi and nucleolar remnants against the dissociation agents used during the extraction procedure [19,22,25]. Numerous studies led to the conclusion that the changes in the pattern of gene expression which occur during cell neoplastic transformation can be intuitively ascribed to the perturbation of interactions between chromatin and proteinaceous NM [26^28]. The precise analysis of protein composition changes which the NM undergoes during cell transformation has been lately reported [29^32]. Thus, NM proteins present in cancer cells but not in their normal counterparts have been described in a numerous organs such as bladder, breast, colon, uterus, prostate and bone [33^39]. In this study we have focused on the protein with a Mr of 36 kDa whose expression is observed in colon, stomach and lung tumor cell nuclei but not in normal tissue [40,41]. By polyclonal antiserum raised against a electrophoretically speci¢c nuclear polypeptide-zone (35^40 kDa) from colon adenocarcinoma (hepatic £exure) we have analyzed the presence of
163
cancer-speci¢c component(s) among nuclear proteins from colorectal tumors appearing in right- and leftsided large intestine as well as in the nuclear fraction of human colon tumor cell lines. Using this antiserum we have veri¢ed the presence of this particular antigen(s) among colonic tumor nuclear fractions: chromatin and NM, and subfractions of the latter, i.e., NMi with nucleolar remnants and NMp . 2. Materials and methods 2.1. Patients Fresh human tumor and normal tissue samples were obtained from 35 patients with colorectal carcinomas undergoing surgical resection at the Surgical Oncology Clinic of Medical University of 6o¨dz¨, Poland. The patients comprised 16 women and 19 men, with mean age 60.4 years (range 18^77 years). The location of the tumor in these patients was as follows: 11 in right-sided (including the cecum, ascending and transverse colon) and 24 in left-sided colon (descending, sigmoid colon and rectum). The tumors were staged according to Dukes classi¢cation. 2.2. Colon tumor cell line cultures Cells HT29 derived from a 44 year old female with colon adenocarcinoma were obtained from the Deutsche Krebsforschungzentrum Heidelberg, Germany. Cells SW620 isolated from a lymph node metastasis of grade III-IV adenocarcinoma of the colon of a 51 year old male were purchased from ECACC (Cat. No. 87051203, Salisbury, UK). Cells were grown in OptiMEM medium supplemented with 5% fetal bovine serum and glutamine (2 mM), at 37³C in 5% CO2 , 95% humidi¢ed air. Cells were passaged after reaching subcon£uency, using 0.5% trypsin/0.02% EDTA solution (all reagents from Gibco, BRL, Life Technologies, UK), and seeded at the density 3U104 cells/ml in 25 ml or 75 ml £asks (Falcon, Becton Dickinson, Heidelberg, Germany). All cells under investigation were screened for Mycoplasma contamination by using Mycoplasma Detection kit enzyme immunoassay (Boehringer Mannheim) and were found to be negative. For the experiment, cells were collected, from
BBADIS 61933 19-5-00
164
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
the full monolayer, by mechanical means (scraping) and washes twice with Ca2 , Mg2 -free PBS saline solution. 2.3. Preparation of nuclei The samples of normal or cancerous material were minced by ¢ne dissection and homogenized (4³C) in 0.25 M sucrose, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM phenylmethylsulfonyl £uoride (PMSF), 50 mM Tris^HCl (pH 7.4), followed by centrifugation of the homogenate at 800Ug for 10 min. The centrifugation was repeated and the crude nuclei was puri¢ed by the sucrose method [42]. 2.4. Isolation of the NM NM preparations were isolated by the modi¢ed method of Belgrader et al. [19]. The nuclei prepared by sucrose technique were washed twice in TM2 bu¡er: 10 mM Tris^HCl, pH 7.4, 2 mM MgCl2 , 1 mM PMSF and subsequently centrifuged at 1000Ug (10 min, 4³C). The nuclei were resuspended in TM2 bu¡er and stabilized by incubation for 1 h at 37³C. MgCl2 was added to the nuclei in TM2 bu¡er to a ¢nal concentration of 5 mM. Then nuclei suspension was digested with DNase I (Sigma) (0.2 mg DNase I/1 mg DNA) for 20 min at 37³C. The digestion was stopped by placing the probe on ice. A stock solution of 2 M ammonium sulfate (AS) was slowly (dropwise) added to a ¢nal concentration of 0.25 M AS. The nuclei suspension was then twice extracted in a glass^te£on homogenizer with 0.25 AS in TM 0.2 bu¡er: 10 mM Tris^HCl, pH 7.4, 0.2 mM MgCl2 , 1 mM PMSF to remove the soluble chromatin proteins, and centrifuged at 2000Ug for 15 min (4³C). The NM pellet was resuspended in TM 0.2 bu¡er and used immediately or stored at 370³C. 2.5. Fractionation of nuclei For fractionation of cell nuclei the combination of Stuurman et al.'s [22] and Payrastre et al.'s [43] techniques were employed. Nuclei prepared by the sucrose method were washed twice in STM bu¡er (0.25 M sucrose, 50 mM Tris^HCl pH 7.4, 5 mM MgCl2 , 1 mM PMSF). For the preparation of NMi nuclei prepared as above were ¢rst stabilized by in-
cubation for 1 h at 37³C with 0.5 mM sodium tetrathionate (NaTT) in STM bu¡er. Nuclei were washed three times with STM and digested with DNase I (0.2 mg enzyme/1 mg DNA) for 20 min at 37³C. AS was added dropwise to a ¢nal concentration of 0.25 M AS. After extraction of AS-soluble chromatin proteins on ice the NM was pelleted at 2000Ug for 10 min. Supernatant containing AS fraction was dialyzed against water (several changes). NM pellet was resuspended and incubated in STM bu¡er containing 0.25 M AS and 40 mM dithiothreitol for 20 min at 37³C. The solubilized NMi was cleared from NMp by centrifugation at 10 000Ug for 10 min. The supernatant was dialyzed against 10 mM ammonium acetate (pH 7.4) with several changes of acetate and stored lyophilized. The NMp was washed with 10 mM Tris^HCl, pH 7.4, 0.2 MgCl2 , 1 mM PMSF and used immediately or stored frozen at 370³C. The pellet of NM before fractionation on NMi and NMp was used for comparison studies. 2.6. Antiserum production Rabbit polyclonal antiserum was raised against electrophoretically speci¢c nuclear polypeptides with a molecular mass of 35^40 kDa separated from adenocarcinoma of colon (hepatic £exure) in the course of preparative electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide slab gel. The polypeptide-zone with an appropriate molecular mass was excised from the gel, homogenized in PBS (145 mM NaCl^10 mM phosphate bu¡er, pH 7.4), combined with the complete Freund's adjuvant (1:1, v/v, Miles Biochem.), and injected intradermally. Thereafter, booster injections were given at weekly intervals for 4 weeks. A total amount of approximately 200 Wg of the antigen was injected intradermally. The rabbits were bled 1 week after the ¢nal injection and the serum was assayed for antibodies using immunoblotting technique. The rabbits were bled prior to the antigen injection, and the serum was used for control studies. 2.7. One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Samples were mixed with 0.9 vol. of solubilizing bu¡er (10% glycerol, 4% SDS, 25 Wg pyronin Y, 125
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
mM Tris^HCl, pH 6.8, 1 mM PMSF), with 0.1 vol. of 2-mercaptoethanol, and heated in boiling water for 5 min. The PAGE was performed as described by Laemmli [44] at 15 mA slab until the pyronin Y marker reached the end of the 3.0% stacking gel and then at 30 mA/slab in the 11.2% resolving gel until the marker dye reached the bottom of the gel. About 20 and 500 Wg of protein were loaded per slot in the case of analytical or preparative electrophoresis, respectively. The slab gels were stained with silver nitrate by the method of Wray et al. [45] or Coomassie brilliant Blue R-250 method [46]. Mr was determined by comparing the mobilities of proteins to those of known molecular mass standards (Sigma). The proteins used as standards were: myosin (205 kDa), L-galactosidase (116 kDa), L-phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and K-lactalbumin (14.2 kDa). 2.8. Two-dimensional PAGE Two-dimensional electrophoresis was carried out according to the method of O'Farrell [47] as modi¢ed by Cupo et al. [48]. The ¢rst-dimensional equilibration isoelectrofocusing gel containing a 2.0% total carrier ampholyte mixture (composed of pH range 4.0^6.5, 6.5^9.0 and 3.0^10.0). The ¢nal acrylamide concentration was 4.0% with a cross-linking of 2.5% N,NP-methylene-bis-acrylamide. The detergent present in the gel was 0.025 M 3-[3-cholamido-propyl]-dimethylammonio-1-propanesulfonate (CHAPS). Proteins were solubilized in 8.0 M urea, 2.0% carrier ampholytes, 0.065 M dithiothreitol, 0.5% Nonidet P-40, 0.065 M CHAPS and samples of about 200 Wg were overlaid on the gels. The gels were electrophoresed for 14 h at 300 V and for 2 h at 400 V. The electrolyte solutions were 0.02 M NaOH (cathode) and 0.01 M H3 PO4 (anode). The pH of the blank gels was measured at 0.5 cm intervals in 0.01 M KCl using a Beckman pH-meter. After isoelectric focusing, the gels were equilibrated for 30 min in 10.0% glycerol, 5.0% 2-mercaptoethanol, 2.3% SDS, and 0.0625 M Tris^HCl at pH 6.8. Electrophoresis in second dimension was carried out on a 8.0% polyacrylamide gel according to Laemmli [44].
165
2.9. Immunoblotting assays Electrophoretically separated proteins were blotted onto Immobilon P according to the method of Towbin et al. [49]. For immunodetection of antigen immobilized on membrane the alkaline-phosphatase technique was performed. The ¢lters were incubated for 1 h at room temperature in 0.5% non-fat dry milk in TBS (10 mM Tris^HCl, pH 7.5, 150 mM NaCl) to saturate the nonspeci¢c protein binding sites. Immobilon P ¢lters were then incubated with primary antiserum at 1:150 dilution in TBS^0.5% non-fat dry milk overnight in cold room. After washing several times in TBS containing 0.05% Tween 20 (TBST) ¢lters were incubated with goat anti-rabbit immunoglobulin conjugated with alkaline phosphatase (Sigma) at 1:6000 dilution in TBS^0.5% non-fat dry milk for 2 h at room temperature. After washing with TBST speci¢c antigen^antibody interactions were visualized by incubation with substrate solution (0.30 mg/ml of nitro blue tetrazolium and 0.15 mg/ml of 5bromo-3-indolyl phosphate in 100 mM Tris^HCl, 100 mM NaCl and 5 mM MgCl2 , pH 9.5), prepared according to the method of Leary et al. [50]. 2.10. Analytical procedures Protein was estimated by the method of Lowry et al. [51] and DNA was determined spectrophotometrically. 3. Results and discussion Epidemiologic and genetic studies suggest that the mechanisms of colorectal tumorigenesis may di¡er according to tumor location [8,11,52,53]. It is worth noting that countries in which the incidence of colonic tumor is high have a relatively higher proportion of left-sided tumors whereas, countries in which the incidence is lower reveal a greater proportion of right-sided tumors. Previous results from our laboratory [40] demonstrated that during colorectal carcinogenesis speci¢c expression of some proteins from four cellular fractions, i.e. nuclear, mitochondrial, microsomal and cytosolic took place. Our interest is focused on nuclear proteins from cancerous and normal colon
BBADIS 61933 19-5-00
166
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
qualitative representation (Fig. 2). The predominant spots in the colon tumor nuclei fraction were observed in the area of two-dimensional gels with Mr / pI of 44^48/5.3^6.0, 58^64/5.3^5.5 and 60^68/5.7^ 7.2; whereas in normal nuclei, with Mr /pI of 44^48/ 5.6^6.3, 50^59/5.3^6.3, 70^85/5.4^6.3 and 115^140/ 5.6^6.2. Some components distributed in the area of gels with low Mr , i.e., of 28^40 kDa as well as with high Mr , i.e., of 65^70 and 120^130 kDa appeared to be speci¢c for malignant tissue. Two spots with a Mr of 36 kDa located in the area of gels corresponding to pI 5.3 and 5.8 were detected only in tumor colon specimens. They represent probably the isoforms of p36 protein. It should be emphasized that the sensitive method of protein detection with
Fig. 1. SDS-PAGE of nuclear proteins from normal (I) and tumor (II) specimens of the anus stained with silver nitrate [45]. The symbol [d] indicates quantitative or qualitative polypeptide di¡erences. Arrows show the positions of molecular mass standards: myosin (205 kDa), L-galactosidase (116 kDa), L-phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and K-lactalbumin (14.2 kDa).
since the diversity in their electrophoretic patterns is expressed to a signi¢cant extent. As it was documented by SDS-PAGE, gel patterns of nuclear proteins from both normal and colorectal cancer samples showed many common constituents. However, some di¡erences mainly among polypeptides with a Mr of 21^30; 30^32; 35^40; 49^54 and 63^69 kDa were observed (Fig. 1). The components with a Mr of 23, 25, 36 and 38 kDa seem to be colorectal cancerassociated. Electrophoretic analysis of nuclear proteins isolated from 35 cancer samples obtained from di¡erent anatomic regions of large intestine show reproducible patterns, with slightly quantitative di¡erences, mainly in the region with a Mr of 35^40 kDa. Since some di¡erences were found between nuclear proteins of normal and colorectal cancer specimens we have extended analysis of their speci¢city by twodimensional PAGE. A comparison of two-dimensional electrophoretic behavior of proteins from normal and tumor nuclear fraction revealed their complexity and diversities in their quantitative and
Fig. 2. High resolution two-dimensional gel electrophoresis of nuclear proteins from normal (I) and tumor (II) specimens of the rectum followed by silver nitrate staining [45]. Isoelectric focusing (IFPA) in 4.0% polyacrylamide is shown on the abscissa, and SDS-PAGE in 8.0% polyacrylamide is shown on the ordinate. The parentheses [] indicate quantitative or qualitative polypeptide di¡erences, and yy two spots of polypeptide with Mr 36 kDa.
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
167
Fig. 3. Immunoblot analysis of nuclear protein fraction from normal (I) and tumor (II) specimens of di¡erent large intestine anatomic regions. (a) Western blot analysis of nuclear proteins from normal and adenocarcinoma of colon (hepatic £exure) in the presence of rabbit serum (dilution 1:150) before immunization (pre-immunized; pI) and after immunization (immunized; I) with nuclear polypeptides with Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). (b) Western analysis of nuclear proteins of normal and tumor specimens obtained from ascending colon (A), sigmoid colon (B), and rectum (C) in the presence of polyclonal antiserum (dilution 1:150) raised against electrophoretically speci¢c nuclear polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). SDS-PAGE of nuclear proteins from normal and tumor specimens of sigmoid colon (BP) stained with Coomassie brilliant Blue R-250 is included. Arrows indicate the position of p36 polypeptide.
silver nitrate often produced spots with shades of yellow or light brown visible on original gels but indistinguishable on the black-and-white photograph (i.e., the components with Mr /pI 28^40/5.3^6.0 in the case of tumor nuclear fraction). Electrophoretic diversity of nuclear proteins from normal and cancerous colon especially with a Mr of 35^40 kDa was further explored by immunological assays. Rabbit polyclonal antiserum was raised against colon adenocarcinoma (hepatic £exure) nuclear polypeptides with a Mr of 35^40 kDa, separated by preparative SDS-PAGE. The speci¢city of obtained serum was veri¢ed by Western blotting technique in the presence of adenocarcinoma and normal colon nuclear proteins and also with preimmune serum (Fig. 3a). Fig. 3b demonstrates the panel of immunoblot assays of nuclear proteins from normal and cancer specimens of di¡erent anatomic regions of large intestine. In Fig. 3b (BP)is shown also an example of SDS-PAGE distribution of nuclear proteins obtained from normal and tumor sigmoid colon followed by Coomassie brilliant Blue R250 staining. It was noticed that obtained antiserum cross-re-
acted with most of the tumor nuclear protein preparations, i.e., 30 of 35 (85.7%) but not with those from normal tissue. The above antiserum recognizes the antigen with a Mr of 36 kDa (p36), and in some cases also a component with a Mr of 32 kDa (p32). Nonspeci¢c cross-reaction with high molecular weight components can be observed sometimes while no cross-reaction with preimmune serum was seen. In studies performed until now p36 nuclear antigen was detected in nine of 11 (81.8%) and 21 of 24 (87.5%) of right- and left-sided tumors, respectively. The frequency of p36 expression in right-sided colon tumors is slightly lower than that observed in leftsided tumors. A higher degree of p36 immunostaining in left-sided tumors is probably caused by the fact that in these anatomic regions of large intestine more aggressive tumors are developed [10,11,52,53]. Our previous data have revealed [41] that the progression of colorectal cancer classi¢ed by Dukes staging was accompanied by the increase of this particular antigen. Since colorectal cancers consist of a usually heterogeneous population of cells, the nuclear fraction was also isolated from tumor cell lines derived from epithelial colon adenocarcinomas, i.e.,
BBADIS 61933 19-5-00
168
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
Fig. 4. Immunoblot (a) and SDS-PAGE (b) analysis of nuclear proteins from tumor cell lines HT29 and SW620. Western blot analysis was done in the presence of polyclonal antiserum raised against electrophoretically speci¢c nuclear polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). Gels after SDS-PAGE were stained with Coomassie brilliant Blue R-250. Arrows indicate the position of p36 polypeptide.
HT29, and SW620 (Fig. 4a,b). It was found that the available serum recognized p36 nuclear antigen in above tumor cell lines, however its concentration showed some quantitative di¡erences. Considerably elevated expression of p36 antigen was observed in nuclear fraction originating from SW620 cell line which is characterized by high potential to form metastasis (Fig. 4a). The obtained data con¢rmed that this particular antigen appears during colorectal tumorigenesis and is not a result of contamination by stromal cells or in£ammatory cell in¢ltration. Nuclear structural alterations are also prevalent in cancerous cells, they are commonly used as markers
of transformation for many types of cancer [12,24,25,33^39]. It is generally accepted that morphologic changes in nuclear shape and size during carcinogenesis may re£ect changes in the protein composition of the NM [12,26^28]. Several laboratories have shown alterations in NM protein expression during normal cell progression toward malignant phenotype [30^39]. Since the NM plays a central role in maintenance of gene expression and chromatin remodeling during transformation of cells we have concentrated on studies of colorectal cancerspeci¢c p36 antigen within nuclear subfractions. In initial experiments we have obtained nuclear matrices from normal and colorectal cancer samples by the modi¢ed method of Belgrader et al. [19] from nuclei incubated for 1 h at 37³C. This method eliminated the use of high salt extraction with 1.6^2.0 M NaCl of histones and other soluble components of cell nuclei during NM isolation. For this purpose it employed a milder extraction agent, i.e., 0.20^0.25 M AS. As it was shown by Western blotting assays the antiserum raised against adenocarcinoma nuclear polypeptides with a Mr of 35^40 kDa stained p36 antigen in 13 of 13 NM preparations isolated from colorectal tumor specimens, but not in any normal colonic samples (Fig. 5a). In further studies we have analyzed in more detail the association of p36 antigen with nuclear subfractions of colorectal tumor samples. By a selective extraction the nuclei can be dissected in the chromatin fraction and the NM. The nuclear subfraction ob-
Fig. 5. Immunoblot analysis of NM (a) and nuclear subfractions (b) from normal and adenocarcinoma of the rectum. (a) Western blot analysis of NM proteins from normal (I) and tumor (II) specimens isolated by Belgrader et al. [19] method. (b) Western blot analysis of nuclear subfractions, i.e., NM, and chromatin (AS) from tumor specimens isolated according to Stuurman et al. [22] and Payrastre et al. [43]. Immunodetection was performed in the presence of polyclonal antiserum (dilution 1:150) raised against electrophoretically speci¢c polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). (c) Electrophoretic pattern of NM, NMi , NMp , and AS nuclear subfractions stained with Coomassie brilliant Blue R-250. Arrows indicate the position of p36 polypeptide.
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
tained during extraction of DNaseI treated puri¢ed nuclei (stabilized for 1h at 37³C) with 0.25 M AS bu¡ered solution constitutes chromatin fraction. The NM can be further separated biochemically into the NMi and the NMp by treatment with reducing agent, i.e. dithiothreitol [22,23,43]. Immunodetection of p36 antigen in obtained colorectal tumor nuclear subfractions is demonstrated in Fig. 5b. Used antiserum stains p36 band in unfractionated NM and slightly also in chromatin fraction. p36 antigen is clearly enriched in the IM but absent in the NMp . This observation seems to be interesting since just this NM substructure is associated with actively transcribed genes and serve as site for processing and transport of heterogeneous nuclear RNA [18,24,54^ 56]. Generally, the NM appears to be fundamentally involved in transcription regulation, which presumably includes alteration in the normal cellular phenotype as a consequence of cancer development. The precise analysis of protein composition changes which the NM undergoes during neoplastic transformation has up to now been limited to only few systems [26,31^39]. In 1994 Keesee et al. [36] ¢rst described six NM proteins expressed in adenocarcinoma of the colon that were absent in normal colonic tissue. Recently, speci¢c NM proteins have been described in colon adenocarcinoma and ulcerative colitis, which is recognized as a pre-neoplastic state of the colon [57,58]. Colorectal cancer-speci¢c p36 antigen detected in our experiments in unfractioned nuclei and dominant in the IM appears to be di¡erent from the above described proteins [36,57,58]. At this time, it is di¤cult to determine its possible function. Expression of this particular polypeptide in NM and also in chromatin suggests the dynamic status of p36 in cell nuclei and possible regulatory function. Expression of p36 antigen may be related to alterations in genetic or molecular signaling pathways, common in the development of colorectal cancer. This protein may be considered as a potential marker for the colorectal tumorigenesis. Obtained results extend similar ¢ndings of cancerassociated NM protein(s) in neoplasms of di¡erent organs [26,33^39,57,58] and suggest that polypeptides of this important nuclear substructure may represent a new cancer biomarker with e¤ciency for the
169
detection and/or in the management of some types of cancer.
References [1] R.Y. Demers, K.C. Parsons, Am. J. Ind. Med. 26 (1994) 33^ 45. [2] P.G. Johnston, C.J. Allegra, Sem. Oncol. 22 (1995) 418^ 432. [3] Z. Wronkowski, Sci. Am. 65 (1997) 93. [4] E.R. Fearon, B. Vogelstein, Science 61 (1990) 759^761. [5] K.W. Kinzler, B. Vogelstein, Cell 87 (1996) 159^170. [6] M. Saito, A. Yamaguchi, T. Goi, T. Tsuchiyama, G. Nakagawara, T. Urano, H. Shiku, K. Furukawa, Oncology 56 (1999) 134^141. [7] X.F. Sun, S. Ru«tten, H. Zhang, B. Nordenskjo«ld, J. Clin. Oncol. 17 (1999) 1745^1750. [8] J. Breivik, G. Gaudernack, Adv. Cancer Res. 76 (1999) 187^ 212. [9] H. Muta, M. Noguchi, M. Perucho, K. Ushio, K. Sugihara, A. Ochiai, H. Nawata, S. Hirohashi, Cancer 77 (1995) 265^ 270. [10] J. Ru«scho¡, W. Dietmaier, J. Lu«ttges, G. Seitz, T. Bocker, Zirngbl, J. Schlegel, H.K. Schackert, K.W. Jauch, F. Hofstaedter, Am. J Pathol. 150 (1997) 1815^1824. [11] M.E. Lleonart, J. Garcia-Foncillas, R. Sanchez-Prieto, P. Martin, A. Moreno, S. Ramon y Cajal, Cancer 83 (1998) 889^895. [12] K.J. Pienta, D.S. Co¡ey, Cancer 68 (1991) 2012^2016. [13] J.-W.R. Mulder, G.J.A. O¡erhaus, E.P. de Freyter, J.J. Floyd, S.E. Kern, B. Vogelstein, S.R. Hamilton, Am. J. Pathol. 141 (1992) 797^804. [14] R. van Driel, D.G. Wansiuk, B. van Steensel, M.A. Grande, W. Schul, L. de Jong, Int. Rev. Cytol. 162A (1995) 151^189. [15] J.R. Davie, J. Cell. Biochem. 62 (1996) 149^154. [16] K.M. Wan, J.A. Nickerson, G. Krockmalnic, S. Penman, Proc. Natl. Acad. Sci. USA 91 (1994) 594^598. [17] H.L. Merriman, A.J. van Wijnen, S. Hiebert, J.P. Bidwell, E. Fey, J. Lian, G.S. Stein, Biochemistry 34 (1995) 13125^ 13132. [18] D.C. He, J.A. Nickerson, S. Penman, J. Cell Biol. 110 (1990) 5690. [19] P. Belgrader, A.J. Siegel, R. Berezney, J. Cell Sci. 98 (1991) 281^291. [20] J.A. Nickerson, G. Krockmalnic, K.M. Wan, S. Penman, Proc. Natl. Acad. Sci. USA 94 (1997) 4446^4450. [21] R. Strick, U.K. Laemmli, Cell 83 (1995) 1137^1148. [22] N. Stuurman, A.M.L. Meijne, A.J. van der Pol, L. de Jong, R. van Driel, J. van Ronaswoude, J. Biol. Chem. 265 (1990) 5460^5465. [23] S.H. Kaufmann, J.H. Shaper, Exp. Cell Res. 155 (1984) 477^ 495. [24] J.A. Nickerson, S. Penman, Cell Biol. Int. Rep. 16 (1992) 811^826.
BBADIS 61933 19-5-00
170
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
[25] L.M. Neri, B.M. Riederer, A. Valmori, S. Capitani, A.M. Martelli, J. Histochem. Cytochem. 45 (1997) 1317^1328. [26] P. Barboro, I. Alberti, P. Sannna, C. Balbi, C. Allera, E. Patrone, Exp. Cell Res. 225 (1996) 315^327. [27] Z. Kilianska, W.M. Krajewska, R. Xie, L. Klyszejko-Stefanowicz, J.-F. Chiu, J. Cell. Biochem. 45 (1991) 303^310. [28] W.M. Krajewska, A. Lipinska, M. Gaczynski, L. KlyszejkoStefanowicz, Int. J. Biochem. 24 (1992) 759^767. [29] A.S. Coutts, J.R. Davie, H. Dotzlaw, L.C. Murphy, J. Cell. Biochem. 63 (1996) 174^184. [30] M. Kallajoki, M. Osborn, Electrophoresis 15 (1994) 520^ 528. [31] S.K. Samuel, T.M. Minish, J.R. Davie, Biochemistry 66 (1997) 9^15. [32] Y. Lakshmanan, E.N.P. Subong, A.W. Partin, J. Urol. 159 (1998) 1354^1358. [33] R.H. Getzenberg, B.R. Vanety, T.A. Oeler, M.M. Quigley, A. Hakam, M.J. Becich, R.R. Bahnson, Cancer Res. 56 (1996) 1690^1694. [34] N. Miyanaga, H. Akaza, S. Ishikawa, M. Ohtani, K. Kawai, K. Koiso, M. Kobayashi, A. Koyama, T. Takahashi, Eur. J. Urol. 31 (1997) 163^168. [35] P.S. Khanuja, J.E. Lehr, H.D. Soule, S.K. Gehani, A.C. Noto, R.Ch. Choudhary, K. Pienta, J. Cancer Res. 53 (1993) 3394^3398. [36] S.K. Keesee, M.D. Meneghini, R.P. Szaro, Y.-J. Wu, Proc. Natl. Acad. Sci. USA 91 (1994) 1913^1916. [37] S.K. Keesee, J. Marchese, A. Meneses, D. Potz, C. GarciaCuellar, R.P. Szaro, G. Solorza, J.G. de la Garzia, W. YingJye, Exp. Cell Res. 244 (1998) 14^25. [38] A.P. Partin, R.H. Getzenberg, M.J. CarMichael, D. Vindivich, J. Yoo, J.I. Epstein, D.S. Co¡ey, Cancer Res. 53 (1993) 744^746. [39] S.I. Dworetzky, E.G. Fey, S. Penman, J.B. Lian, J.L. Stein, G.S. Stein, Proc. Natl. Acad. Sci. USA 87 (1990) 4605^4609. [40] P. Szymczyk, W.M. Krajewska, J. Jakubik, A. Berner, J. Janczukowicz, U. Mikulska, J. Berner, Z.M. Kilianska, Tumori 82 (1996) 376^381.
[41] P. Szymczyk, W.M. Krajewska, J. Jakubik, A. Berner, J. Berner, M. Brocki, B. Rajczyk, Z.M. Kilianska, Cell Mol. Biol. Lett. 1 (1996) 363^371. [42] G. Blobel, V.R. Potter, Science 154 (1966) 1662^1665. [43] B. Payrastre, M. Nievers, J. Boonstra, M. Breton, A.J. Verkleij, P.M.P. van Bergen, J.P.M. Van Bergenen Henegouwen, J. Biol. Chem. 267 (1992) 5078^5084. [44] U.K. Laemmli, Nature 227 (1970) 680^685. [45] W. Wray, T. Boulikas, V. Wray, R. Hancock, Anal. Biochem. 118 (1982) 197^203. [46] G. Fairbanks, T.J. Steck, D.H.G. Wallach, Biochemistry 10 (1971) 2606^2612. [47] P.H.J. O'Farrell, J. Biol. Chem. 250 (1975) 4007^4021. [48] J.F. Cupo, G.P. Lidgard, W.F. Lichtman, Electrophoresis 11 (1990) 500^504. [49] H. Towbin, T. Staechelin, J. Gordon, Proc. Natl. Acad. Sci. USA 76 (1979) 4350^4354. [50] J.J. Leary, J.J. Brigati, D.C. Ward, Proc. Natl. Acad. Sci. USA 80 (1983) 4045^4049. [51] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265^275. [52] M. Watatani, T. Yoshida, K. Kuroda, S. Ieda, M. Yasutumi, Cancer 77 (1996) 1688^1693. [53] H.T. Lynch, T.C. Smyrk, P. Watson, S.J. Lanspa, J.F. Lynch, P.M. Lynch, J.R. Cavalieri, C.R. Boland, Gastroenterology 104 (1993) 1535^1549. [54] Y. Xing, C.V. Johnson, R.P. Dobner, J.B. Lawrence, Science 259 (1993) 1326^1330. [55] B. Chabot, S. Bisotto, M. Vincent, Nucleic Acids Res. 23 (1995) 3206^3213. [56] Ch.M. Clemson, J.A. McNeil, H.F. Willard, J.B. Lawrence, J. Cell Biol. 132 (1996) 259^275. [57] R.S. Izzo, C. Pellecchia, Scand. J. Gastroenterol. 33 (1998) 191^194. [58] R.S. Izzo, C. Pellecchia, Biochem. Mol. Biol. Int. 40 (1996) 521^526.
BBADIS 61933 19-5-00
Colorectal cancer-associated nuclear antigen Piotr Szymczyk a , JarosIaw Jakubik b , Wanda M. Krajewska a , Danuta Dus¨ c , Jan Berner b , Zo¢a M. Kilian¨ska a; * a
Department of Cytobiochemistry, University of 6o¨dz¨, ul. Banacha 12/16, 90-237 6o¨dz¨, Poland Department of Surgical Oncology, Medical University of 6o¨dz¨, ul. Paderewskiego 4, 93-509 6o¨dz¨, Poland Institute of Immunology and Experimental Therapy, Polish Academy of Science, ul. Weigla 12, 53-114 WrocIaw, Poland b
c
Received 28 April 1999; received in revised form 15 February 2000; accepted 29 February 2000
Abstract By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with Mr of 35^40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Colorectal cancer; Colon tumor cell line; Tumor marker; Nuclear matrix; Chromatin
1. Introduction Colorectal cancer is a common disease in the industrialized Western Europe countries and the United States [1,2]. This cancer is the second leading cause of malignant deaths in Poland [3]. The pathogenesis of colorectal cancer is of particular interest for the complex and only partially understood interaction between environmental factors and genetic background. The current model for colorectal carcinogenesis postulates a multistage progression involving an accumulation of gene mutations (APC, DCC, K-ras, p53, DNA mismatch repair genes), alterations
* Corresponding author. Fax: +48-42-635-44-84.
in gene expression (c-myc, TGFL receptor), and chromosome losses, during which regulation of cell growth is disrupted [4^8]. Recently published data indicate that mechanisms of colorectal tumorigenesis may di¡er according to tumor location [9^11]. It is an accepted opinion that cancers in the proximal colon are associated with intrinsic factors such as bile acids and sex hormones, whereas distal cancers are more closely related to environmental factors such as diet, cigarette smoking, and alcohol consumption [8]. A cellular hallmark of the transformed phenotype is an abnormal nuclear shape [12,13]. Nuclear structural alterations are commonly used as pathological markers in transformation in many types of cancer. Nuclear shape is thought to re£ect the internal nu-
0925-4439 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 0 ) 0 0 0 1 7 - X
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
clear structure and processes, and is determined mainly by the nuclear matrix (NM). This structure organizes DNA into structurally and functionally independent domains [14,15]. Since this structure plays an essential role in supporting most of nuclear functions, i.e. replication, transcription, RNA metabolism and transport, it has been postulated that during carcinogenesis its components would be altered [16,17]. The NM is the nonchromatin structural protein and ribonucleoprotein network that is resistant to nucleases, detergents and high-ionic strength bu¡ers [14,15,18,19]. The resulting structures which retain the overall shape of nuclei, are mainly composed of non-histone proteins and variable amounts of RNA and DNA [18^24]. Ultrastructural investigations have revealed that the NM can be divided into two compartments, i.e. internal matrix (internal network with residual nucleoli described as: NMi ) which contains residual nucleoli, and peripheral matrix (residual lamina-pore complex: NMp ) composed of residual lamina^pore complex [19,23,24]. It has shown that morphological and functional properties of the NM vary depending on the manner employed for its isolation [19,22^24]. Several studies have shown that an incubation of isolated nuclei near physiological temperature of 37³C stabilizes the NMi and nucleolar remnants against the dissociation agents used during the extraction procedure [19,22,25]. Numerous studies led to the conclusion that the changes in the pattern of gene expression which occur during cell neoplastic transformation can be intuitively ascribed to the perturbation of interactions between chromatin and proteinaceous NM [26^28]. The precise analysis of protein composition changes which the NM undergoes during cell transformation has been lately reported [29^32]. Thus, NM proteins present in cancer cells but not in their normal counterparts have been described in a numerous organs such as bladder, breast, colon, uterus, prostate and bone [33^39]. In this study we have focused on the protein with a Mr of 36 kDa whose expression is observed in colon, stomach and lung tumor cell nuclei but not in normal tissue [40,41]. By polyclonal antiserum raised against a electrophoretically speci¢c nuclear polypeptide-zone (35^40 kDa) from colon adenocarcinoma (hepatic £exure) we have analyzed the presence of
163
cancer-speci¢c component(s) among nuclear proteins from colorectal tumors appearing in right- and leftsided large intestine as well as in the nuclear fraction of human colon tumor cell lines. Using this antiserum we have veri¢ed the presence of this particular antigen(s) among colonic tumor nuclear fractions: chromatin and NM, and subfractions of the latter, i.e., NMi with nucleolar remnants and NMp . 2. Materials and methods 2.1. Patients Fresh human tumor and normal tissue samples were obtained from 35 patients with colorectal carcinomas undergoing surgical resection at the Surgical Oncology Clinic of Medical University of 6o¨dz¨, Poland. The patients comprised 16 women and 19 men, with mean age 60.4 years (range 18^77 years). The location of the tumor in these patients was as follows: 11 in right-sided (including the cecum, ascending and transverse colon) and 24 in left-sided colon (descending, sigmoid colon and rectum). The tumors were staged according to Dukes classi¢cation. 2.2. Colon tumor cell line cultures Cells HT29 derived from a 44 year old female with colon adenocarcinoma were obtained from the Deutsche Krebsforschungzentrum Heidelberg, Germany. Cells SW620 isolated from a lymph node metastasis of grade III-IV adenocarcinoma of the colon of a 51 year old male were purchased from ECACC (Cat. No. 87051203, Salisbury, UK). Cells were grown in OptiMEM medium supplemented with 5% fetal bovine serum and glutamine (2 mM), at 37³C in 5% CO2 , 95% humidi¢ed air. Cells were passaged after reaching subcon£uency, using 0.5% trypsin/0.02% EDTA solution (all reagents from Gibco, BRL, Life Technologies, UK), and seeded at the density 3U104 cells/ml in 25 ml or 75 ml £asks (Falcon, Becton Dickinson, Heidelberg, Germany). All cells under investigation were screened for Mycoplasma contamination by using Mycoplasma Detection kit enzyme immunoassay (Boehringer Mannheim) and were found to be negative. For the experiment, cells were collected, from
BBADIS 61933 19-5-00
164
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
the full monolayer, by mechanical means (scraping) and washes twice with Ca2 , Mg2 -free PBS saline solution. 2.3. Preparation of nuclei The samples of normal or cancerous material were minced by ¢ne dissection and homogenized (4³C) in 0.25 M sucrose, 5 mM MgCl2 , 0.5% Triton X-100, 1 mM phenylmethylsulfonyl £uoride (PMSF), 50 mM Tris^HCl (pH 7.4), followed by centrifugation of the homogenate at 800Ug for 10 min. The centrifugation was repeated and the crude nuclei was puri¢ed by the sucrose method [42]. 2.4. Isolation of the NM NM preparations were isolated by the modi¢ed method of Belgrader et al. [19]. The nuclei prepared by sucrose technique were washed twice in TM2 bu¡er: 10 mM Tris^HCl, pH 7.4, 2 mM MgCl2 , 1 mM PMSF and subsequently centrifuged at 1000Ug (10 min, 4³C). The nuclei were resuspended in TM2 bu¡er and stabilized by incubation for 1 h at 37³C. MgCl2 was added to the nuclei in TM2 bu¡er to a ¢nal concentration of 5 mM. Then nuclei suspension was digested with DNase I (Sigma) (0.2 mg DNase I/1 mg DNA) for 20 min at 37³C. The digestion was stopped by placing the probe on ice. A stock solution of 2 M ammonium sulfate (AS) was slowly (dropwise) added to a ¢nal concentration of 0.25 M AS. The nuclei suspension was then twice extracted in a glass^te£on homogenizer with 0.25 AS in TM 0.2 bu¡er: 10 mM Tris^HCl, pH 7.4, 0.2 mM MgCl2 , 1 mM PMSF to remove the soluble chromatin proteins, and centrifuged at 2000Ug for 15 min (4³C). The NM pellet was resuspended in TM 0.2 bu¡er and used immediately or stored at 370³C. 2.5. Fractionation of nuclei For fractionation of cell nuclei the combination of Stuurman et al.'s [22] and Payrastre et al.'s [43] techniques were employed. Nuclei prepared by the sucrose method were washed twice in STM bu¡er (0.25 M sucrose, 50 mM Tris^HCl pH 7.4, 5 mM MgCl2 , 1 mM PMSF). For the preparation of NMi nuclei prepared as above were ¢rst stabilized by in-
cubation for 1 h at 37³C with 0.5 mM sodium tetrathionate (NaTT) in STM bu¡er. Nuclei were washed three times with STM and digested with DNase I (0.2 mg enzyme/1 mg DNA) for 20 min at 37³C. AS was added dropwise to a ¢nal concentration of 0.25 M AS. After extraction of AS-soluble chromatin proteins on ice the NM was pelleted at 2000Ug for 10 min. Supernatant containing AS fraction was dialyzed against water (several changes). NM pellet was resuspended and incubated in STM bu¡er containing 0.25 M AS and 40 mM dithiothreitol for 20 min at 37³C. The solubilized NMi was cleared from NMp by centrifugation at 10 000Ug for 10 min. The supernatant was dialyzed against 10 mM ammonium acetate (pH 7.4) with several changes of acetate and stored lyophilized. The NMp was washed with 10 mM Tris^HCl, pH 7.4, 0.2 MgCl2 , 1 mM PMSF and used immediately or stored frozen at 370³C. The pellet of NM before fractionation on NMi and NMp was used for comparison studies. 2.6. Antiserum production Rabbit polyclonal antiserum was raised against electrophoretically speci¢c nuclear polypeptides with a molecular mass of 35^40 kDa separated from adenocarcinoma of colon (hepatic £exure) in the course of preparative electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide slab gel. The polypeptide-zone with an appropriate molecular mass was excised from the gel, homogenized in PBS (145 mM NaCl^10 mM phosphate bu¡er, pH 7.4), combined with the complete Freund's adjuvant (1:1, v/v, Miles Biochem.), and injected intradermally. Thereafter, booster injections were given at weekly intervals for 4 weeks. A total amount of approximately 200 Wg of the antigen was injected intradermally. The rabbits were bled 1 week after the ¢nal injection and the serum was assayed for antibodies using immunoblotting technique. The rabbits were bled prior to the antigen injection, and the serum was used for control studies. 2.7. One-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Samples were mixed with 0.9 vol. of solubilizing bu¡er (10% glycerol, 4% SDS, 25 Wg pyronin Y, 125
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
mM Tris^HCl, pH 6.8, 1 mM PMSF), with 0.1 vol. of 2-mercaptoethanol, and heated in boiling water for 5 min. The PAGE was performed as described by Laemmli [44] at 15 mA slab until the pyronin Y marker reached the end of the 3.0% stacking gel and then at 30 mA/slab in the 11.2% resolving gel until the marker dye reached the bottom of the gel. About 20 and 500 Wg of protein were loaded per slot in the case of analytical or preparative electrophoresis, respectively. The slab gels were stained with silver nitrate by the method of Wray et al. [45] or Coomassie brilliant Blue R-250 method [46]. Mr was determined by comparing the mobilities of proteins to those of known molecular mass standards (Sigma). The proteins used as standards were: myosin (205 kDa), L-galactosidase (116 kDa), L-phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and K-lactalbumin (14.2 kDa). 2.8. Two-dimensional PAGE Two-dimensional electrophoresis was carried out according to the method of O'Farrell [47] as modi¢ed by Cupo et al. [48]. The ¢rst-dimensional equilibration isoelectrofocusing gel containing a 2.0% total carrier ampholyte mixture (composed of pH range 4.0^6.5, 6.5^9.0 and 3.0^10.0). The ¢nal acrylamide concentration was 4.0% with a cross-linking of 2.5% N,NP-methylene-bis-acrylamide. The detergent present in the gel was 0.025 M 3-[3-cholamido-propyl]-dimethylammonio-1-propanesulfonate (CHAPS). Proteins were solubilized in 8.0 M urea, 2.0% carrier ampholytes, 0.065 M dithiothreitol, 0.5% Nonidet P-40, 0.065 M CHAPS and samples of about 200 Wg were overlaid on the gels. The gels were electrophoresed for 14 h at 300 V and for 2 h at 400 V. The electrolyte solutions were 0.02 M NaOH (cathode) and 0.01 M H3 PO4 (anode). The pH of the blank gels was measured at 0.5 cm intervals in 0.01 M KCl using a Beckman pH-meter. After isoelectric focusing, the gels were equilibrated for 30 min in 10.0% glycerol, 5.0% 2-mercaptoethanol, 2.3% SDS, and 0.0625 M Tris^HCl at pH 6.8. Electrophoresis in second dimension was carried out on a 8.0% polyacrylamide gel according to Laemmli [44].
165
2.9. Immunoblotting assays Electrophoretically separated proteins were blotted onto Immobilon P according to the method of Towbin et al. [49]. For immunodetection of antigen immobilized on membrane the alkaline-phosphatase technique was performed. The ¢lters were incubated for 1 h at room temperature in 0.5% non-fat dry milk in TBS (10 mM Tris^HCl, pH 7.5, 150 mM NaCl) to saturate the nonspeci¢c protein binding sites. Immobilon P ¢lters were then incubated with primary antiserum at 1:150 dilution in TBS^0.5% non-fat dry milk overnight in cold room. After washing several times in TBS containing 0.05% Tween 20 (TBST) ¢lters were incubated with goat anti-rabbit immunoglobulin conjugated with alkaline phosphatase (Sigma) at 1:6000 dilution in TBS^0.5% non-fat dry milk for 2 h at room temperature. After washing with TBST speci¢c antigen^antibody interactions were visualized by incubation with substrate solution (0.30 mg/ml of nitro blue tetrazolium and 0.15 mg/ml of 5bromo-3-indolyl phosphate in 100 mM Tris^HCl, 100 mM NaCl and 5 mM MgCl2 , pH 9.5), prepared according to the method of Leary et al. [50]. 2.10. Analytical procedures Protein was estimated by the method of Lowry et al. [51] and DNA was determined spectrophotometrically. 3. Results and discussion Epidemiologic and genetic studies suggest that the mechanisms of colorectal tumorigenesis may di¡er according to tumor location [8,11,52,53]. It is worth noting that countries in which the incidence of colonic tumor is high have a relatively higher proportion of left-sided tumors whereas, countries in which the incidence is lower reveal a greater proportion of right-sided tumors. Previous results from our laboratory [40] demonstrated that during colorectal carcinogenesis speci¢c expression of some proteins from four cellular fractions, i.e. nuclear, mitochondrial, microsomal and cytosolic took place. Our interest is focused on nuclear proteins from cancerous and normal colon
BBADIS 61933 19-5-00
166
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
qualitative representation (Fig. 2). The predominant spots in the colon tumor nuclei fraction were observed in the area of two-dimensional gels with Mr / pI of 44^48/5.3^6.0, 58^64/5.3^5.5 and 60^68/5.7^ 7.2; whereas in normal nuclei, with Mr /pI of 44^48/ 5.6^6.3, 50^59/5.3^6.3, 70^85/5.4^6.3 and 115^140/ 5.6^6.2. Some components distributed in the area of gels with low Mr , i.e., of 28^40 kDa as well as with high Mr , i.e., of 65^70 and 120^130 kDa appeared to be speci¢c for malignant tissue. Two spots with a Mr of 36 kDa located in the area of gels corresponding to pI 5.3 and 5.8 were detected only in tumor colon specimens. They represent probably the isoforms of p36 protein. It should be emphasized that the sensitive method of protein detection with
Fig. 1. SDS-PAGE of nuclear proteins from normal (I) and tumor (II) specimens of the anus stained with silver nitrate [45]. The symbol [d] indicates quantitative or qualitative polypeptide di¡erences. Arrows show the positions of molecular mass standards: myosin (205 kDa), L-galactosidase (116 kDa), L-phosphorylase (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and K-lactalbumin (14.2 kDa).
since the diversity in their electrophoretic patterns is expressed to a signi¢cant extent. As it was documented by SDS-PAGE, gel patterns of nuclear proteins from both normal and colorectal cancer samples showed many common constituents. However, some di¡erences mainly among polypeptides with a Mr of 21^30; 30^32; 35^40; 49^54 and 63^69 kDa were observed (Fig. 1). The components with a Mr of 23, 25, 36 and 38 kDa seem to be colorectal cancerassociated. Electrophoretic analysis of nuclear proteins isolated from 35 cancer samples obtained from di¡erent anatomic regions of large intestine show reproducible patterns, with slightly quantitative di¡erences, mainly in the region with a Mr of 35^40 kDa. Since some di¡erences were found between nuclear proteins of normal and colorectal cancer specimens we have extended analysis of their speci¢city by twodimensional PAGE. A comparison of two-dimensional electrophoretic behavior of proteins from normal and tumor nuclear fraction revealed their complexity and diversities in their quantitative and
Fig. 2. High resolution two-dimensional gel electrophoresis of nuclear proteins from normal (I) and tumor (II) specimens of the rectum followed by silver nitrate staining [45]. Isoelectric focusing (IFPA) in 4.0% polyacrylamide is shown on the abscissa, and SDS-PAGE in 8.0% polyacrylamide is shown on the ordinate. The parentheses [] indicate quantitative or qualitative polypeptide di¡erences, and yy two spots of polypeptide with Mr 36 kDa.
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
167
Fig. 3. Immunoblot analysis of nuclear protein fraction from normal (I) and tumor (II) specimens of di¡erent large intestine anatomic regions. (a) Western blot analysis of nuclear proteins from normal and adenocarcinoma of colon (hepatic £exure) in the presence of rabbit serum (dilution 1:150) before immunization (pre-immunized; pI) and after immunization (immunized; I) with nuclear polypeptides with Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). (b) Western analysis of nuclear proteins of normal and tumor specimens obtained from ascending colon (A), sigmoid colon (B), and rectum (C) in the presence of polyclonal antiserum (dilution 1:150) raised against electrophoretically speci¢c nuclear polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). SDS-PAGE of nuclear proteins from normal and tumor specimens of sigmoid colon (BP) stained with Coomassie brilliant Blue R-250 is included. Arrows indicate the position of p36 polypeptide.
silver nitrate often produced spots with shades of yellow or light brown visible on original gels but indistinguishable on the black-and-white photograph (i.e., the components with Mr /pI 28^40/5.3^6.0 in the case of tumor nuclear fraction). Electrophoretic diversity of nuclear proteins from normal and cancerous colon especially with a Mr of 35^40 kDa was further explored by immunological assays. Rabbit polyclonal antiserum was raised against colon adenocarcinoma (hepatic £exure) nuclear polypeptides with a Mr of 35^40 kDa, separated by preparative SDS-PAGE. The speci¢city of obtained serum was veri¢ed by Western blotting technique in the presence of adenocarcinoma and normal colon nuclear proteins and also with preimmune serum (Fig. 3a). Fig. 3b demonstrates the panel of immunoblot assays of nuclear proteins from normal and cancer specimens of di¡erent anatomic regions of large intestine. In Fig. 3b (BP)is shown also an example of SDS-PAGE distribution of nuclear proteins obtained from normal and tumor sigmoid colon followed by Coomassie brilliant Blue R250 staining. It was noticed that obtained antiserum cross-re-
acted with most of the tumor nuclear protein preparations, i.e., 30 of 35 (85.7%) but not with those from normal tissue. The above antiserum recognizes the antigen with a Mr of 36 kDa (p36), and in some cases also a component with a Mr of 32 kDa (p32). Nonspeci¢c cross-reaction with high molecular weight components can be observed sometimes while no cross-reaction with preimmune serum was seen. In studies performed until now p36 nuclear antigen was detected in nine of 11 (81.8%) and 21 of 24 (87.5%) of right- and left-sided tumors, respectively. The frequency of p36 expression in right-sided colon tumors is slightly lower than that observed in leftsided tumors. A higher degree of p36 immunostaining in left-sided tumors is probably caused by the fact that in these anatomic regions of large intestine more aggressive tumors are developed [10,11,52,53]. Our previous data have revealed [41] that the progression of colorectal cancer classi¢ed by Dukes staging was accompanied by the increase of this particular antigen. Since colorectal cancers consist of a usually heterogeneous population of cells, the nuclear fraction was also isolated from tumor cell lines derived from epithelial colon adenocarcinomas, i.e.,
BBADIS 61933 19-5-00
168
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
Fig. 4. Immunoblot (a) and SDS-PAGE (b) analysis of nuclear proteins from tumor cell lines HT29 and SW620. Western blot analysis was done in the presence of polyclonal antiserum raised against electrophoretically speci¢c nuclear polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). Gels after SDS-PAGE were stained with Coomassie brilliant Blue R-250. Arrows indicate the position of p36 polypeptide.
HT29, and SW620 (Fig. 4a,b). It was found that the available serum recognized p36 nuclear antigen in above tumor cell lines, however its concentration showed some quantitative di¡erences. Considerably elevated expression of p36 antigen was observed in nuclear fraction originating from SW620 cell line which is characterized by high potential to form metastasis (Fig. 4a). The obtained data con¢rmed that this particular antigen appears during colorectal tumorigenesis and is not a result of contamination by stromal cells or in£ammatory cell in¢ltration. Nuclear structural alterations are also prevalent in cancerous cells, they are commonly used as markers
of transformation for many types of cancer [12,24,25,33^39]. It is generally accepted that morphologic changes in nuclear shape and size during carcinogenesis may re£ect changes in the protein composition of the NM [12,26^28]. Several laboratories have shown alterations in NM protein expression during normal cell progression toward malignant phenotype [30^39]. Since the NM plays a central role in maintenance of gene expression and chromatin remodeling during transformation of cells we have concentrated on studies of colorectal cancerspeci¢c p36 antigen within nuclear subfractions. In initial experiments we have obtained nuclear matrices from normal and colorectal cancer samples by the modi¢ed method of Belgrader et al. [19] from nuclei incubated for 1 h at 37³C. This method eliminated the use of high salt extraction with 1.6^2.0 M NaCl of histones and other soluble components of cell nuclei during NM isolation. For this purpose it employed a milder extraction agent, i.e., 0.20^0.25 M AS. As it was shown by Western blotting assays the antiserum raised against adenocarcinoma nuclear polypeptides with a Mr of 35^40 kDa stained p36 antigen in 13 of 13 NM preparations isolated from colorectal tumor specimens, but not in any normal colonic samples (Fig. 5a). In further studies we have analyzed in more detail the association of p36 antigen with nuclear subfractions of colorectal tumor samples. By a selective extraction the nuclei can be dissected in the chromatin fraction and the NM. The nuclear subfraction ob-
Fig. 5. Immunoblot analysis of NM (a) and nuclear subfractions (b) from normal and adenocarcinoma of the rectum. (a) Western blot analysis of NM proteins from normal (I) and tumor (II) specimens isolated by Belgrader et al. [19] method. (b) Western blot analysis of nuclear subfractions, i.e., NM, and chromatin (AS) from tumor specimens isolated according to Stuurman et al. [22] and Payrastre et al. [43]. Immunodetection was performed in the presence of polyclonal antiserum (dilution 1:150) raised against electrophoretically speci¢c polypeptides with a Mr of 35^40 kDa from adenocarcinoma of colon (hepatic £exure). (c) Electrophoretic pattern of NM, NMi , NMp , and AS nuclear subfractions stained with Coomassie brilliant Blue R-250. Arrows indicate the position of p36 polypeptide.
BBADIS 61933 19-5-00
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
tained during extraction of DNaseI treated puri¢ed nuclei (stabilized for 1h at 37³C) with 0.25 M AS bu¡ered solution constitutes chromatin fraction. The NM can be further separated biochemically into the NMi and the NMp by treatment with reducing agent, i.e. dithiothreitol [22,23,43]. Immunodetection of p36 antigen in obtained colorectal tumor nuclear subfractions is demonstrated in Fig. 5b. Used antiserum stains p36 band in unfractionated NM and slightly also in chromatin fraction. p36 antigen is clearly enriched in the IM but absent in the NMp . This observation seems to be interesting since just this NM substructure is associated with actively transcribed genes and serve as site for processing and transport of heterogeneous nuclear RNA [18,24,54^ 56]. Generally, the NM appears to be fundamentally involved in transcription regulation, which presumably includes alteration in the normal cellular phenotype as a consequence of cancer development. The precise analysis of protein composition changes which the NM undergoes during neoplastic transformation has up to now been limited to only few systems [26,31^39]. In 1994 Keesee et al. [36] ¢rst described six NM proteins expressed in adenocarcinoma of the colon that were absent in normal colonic tissue. Recently, speci¢c NM proteins have been described in colon adenocarcinoma and ulcerative colitis, which is recognized as a pre-neoplastic state of the colon [57,58]. Colorectal cancer-speci¢c p36 antigen detected in our experiments in unfractioned nuclei and dominant in the IM appears to be di¡erent from the above described proteins [36,57,58]. At this time, it is di¤cult to determine its possible function. Expression of this particular polypeptide in NM and also in chromatin suggests the dynamic status of p36 in cell nuclei and possible regulatory function. Expression of p36 antigen may be related to alterations in genetic or molecular signaling pathways, common in the development of colorectal cancer. This protein may be considered as a potential marker for the colorectal tumorigenesis. Obtained results extend similar ¢ndings of cancerassociated NM protein(s) in neoplasms of di¡erent organs [26,33^39,57,58] and suggest that polypeptides of this important nuclear substructure may represent a new cancer biomarker with e¤ciency for the
169
detection and/or in the management of some types of cancer.
References [1] R.Y. Demers, K.C. Parsons, Am. J. Ind. Med. 26 (1994) 33^ 45. [2] P.G. Johnston, C.J. Allegra, Sem. Oncol. 22 (1995) 418^ 432. [3] Z. Wronkowski, Sci. Am. 65 (1997) 93. [4] E.R. Fearon, B. Vogelstein, Science 61 (1990) 759^761. [5] K.W. Kinzler, B. Vogelstein, Cell 87 (1996) 159^170. [6] M. Saito, A. Yamaguchi, T. Goi, T. Tsuchiyama, G. Nakagawara, T. Urano, H. Shiku, K. Furukawa, Oncology 56 (1999) 134^141. [7] X.F. Sun, S. Ru«tten, H. Zhang, B. Nordenskjo«ld, J. Clin. Oncol. 17 (1999) 1745^1750. [8] J. Breivik, G. Gaudernack, Adv. Cancer Res. 76 (1999) 187^ 212. [9] H. Muta, M. Noguchi, M. Perucho, K. Ushio, K. Sugihara, A. Ochiai, H. Nawata, S. Hirohashi, Cancer 77 (1995) 265^ 270. [10] J. Ru«scho¡, W. Dietmaier, J. Lu«ttges, G. Seitz, T. Bocker, Zirngbl, J. Schlegel, H.K. Schackert, K.W. Jauch, F. Hofstaedter, Am. J Pathol. 150 (1997) 1815^1824. [11] M.E. Lleonart, J. Garcia-Foncillas, R. Sanchez-Prieto, P. Martin, A. Moreno, S. Ramon y Cajal, Cancer 83 (1998) 889^895. [12] K.J. Pienta, D.S. Co¡ey, Cancer 68 (1991) 2012^2016. [13] J.-W.R. Mulder, G.J.A. O¡erhaus, E.P. de Freyter, J.J. Floyd, S.E. Kern, B. Vogelstein, S.R. Hamilton, Am. J. Pathol. 141 (1992) 797^804. [14] R. van Driel, D.G. Wansiuk, B. van Steensel, M.A. Grande, W. Schul, L. de Jong, Int. Rev. Cytol. 162A (1995) 151^189. [15] J.R. Davie, J. Cell. Biochem. 62 (1996) 149^154. [16] K.M. Wan, J.A. Nickerson, G. Krockmalnic, S. Penman, Proc. Natl. Acad. Sci. USA 91 (1994) 594^598. [17] H.L. Merriman, A.J. van Wijnen, S. Hiebert, J.P. Bidwell, E. Fey, J. Lian, G.S. Stein, Biochemistry 34 (1995) 13125^ 13132. [18] D.C. He, J.A. Nickerson, S. Penman, J. Cell Biol. 110 (1990) 5690. [19] P. Belgrader, A.J. Siegel, R. Berezney, J. Cell Sci. 98 (1991) 281^291. [20] J.A. Nickerson, G. Krockmalnic, K.M. Wan, S. Penman, Proc. Natl. Acad. Sci. USA 94 (1997) 4446^4450. [21] R. Strick, U.K. Laemmli, Cell 83 (1995) 1137^1148. [22] N. Stuurman, A.M.L. Meijne, A.J. van der Pol, L. de Jong, R. van Driel, J. van Ronaswoude, J. Biol. Chem. 265 (1990) 5460^5465. [23] S.H. Kaufmann, J.H. Shaper, Exp. Cell Res. 155 (1984) 477^ 495. [24] J.A. Nickerson, S. Penman, Cell Biol. Int. Rep. 16 (1992) 811^826.
BBADIS 61933 19-5-00
170
P. Szymczyk et al. / Biochimica et Biophysica Acta 1501 (2000) 162^170
[25] L.M. Neri, B.M. Riederer, A. Valmori, S. Capitani, A.M. Martelli, J. Histochem. Cytochem. 45 (1997) 1317^1328. [26] P. Barboro, I. Alberti, P. Sannna, C. Balbi, C. Allera, E. Patrone, Exp. Cell Res. 225 (1996) 315^327. [27] Z. Kilianska, W.M. Krajewska, R. Xie, L. Klyszejko-Stefanowicz, J.-F. Chiu, J. Cell. Biochem. 45 (1991) 303^310. [28] W.M. Krajewska, A. Lipinska, M. Gaczynski, L. KlyszejkoStefanowicz, Int. J. Biochem. 24 (1992) 759^767. [29] A.S. Coutts, J.R. Davie, H. Dotzlaw, L.C. Murphy, J. Cell. Biochem. 63 (1996) 174^184. [30] M. Kallajoki, M. Osborn, Electrophoresis 15 (1994) 520^ 528. [31] S.K. Samuel, T.M. Minish, J.R. Davie, Biochemistry 66 (1997) 9^15. [32] Y. Lakshmanan, E.N.P. Subong, A.W. Partin, J. Urol. 159 (1998) 1354^1358. [33] R.H. Getzenberg, B.R. Vanety, T.A. Oeler, M.M. Quigley, A. Hakam, M.J. Becich, R.R. Bahnson, Cancer Res. 56 (1996) 1690^1694. [34] N. Miyanaga, H. Akaza, S. Ishikawa, M. Ohtani, K. Kawai, K. Koiso, M. Kobayashi, A. Koyama, T. Takahashi, Eur. J. Urol. 31 (1997) 163^168. [35] P.S. Khanuja, J.E. Lehr, H.D. Soule, S.K. Gehani, A.C. Noto, R.Ch. Choudhary, K. Pienta, J. Cancer Res. 53 (1993) 3394^3398. [36] S.K. Keesee, M.D. Meneghini, R.P. Szaro, Y.-J. Wu, Proc. Natl. Acad. Sci. USA 91 (1994) 1913^1916. [37] S.K. Keesee, J. Marchese, A. Meneses, D. Potz, C. GarciaCuellar, R.P. Szaro, G. Solorza, J.G. de la Garzia, W. YingJye, Exp. Cell Res. 244 (1998) 14^25. [38] A.P. Partin, R.H. Getzenberg, M.J. CarMichael, D. Vindivich, J. Yoo, J.I. Epstein, D.S. Co¡ey, Cancer Res. 53 (1993) 744^746. [39] S.I. Dworetzky, E.G. Fey, S. Penman, J.B. Lian, J.L. Stein, G.S. Stein, Proc. Natl. Acad. Sci. USA 87 (1990) 4605^4609. [40] P. Szymczyk, W.M. Krajewska, J. Jakubik, A. Berner, J. Janczukowicz, U. Mikulska, J. Berner, Z.M. Kilianska, Tumori 82 (1996) 376^381.
[41] P. Szymczyk, W.M. Krajewska, J. Jakubik, A. Berner, J. Berner, M. Brocki, B. Rajczyk, Z.M. Kilianska, Cell Mol. Biol. Lett. 1 (1996) 363^371. [42] G. Blobel, V.R. Potter, Science 154 (1966) 1662^1665. [43] B. Payrastre, M. Nievers, J. Boonstra, M. Breton, A.J. Verkleij, P.M.P. van Bergen, J.P.M. Van Bergenen Henegouwen, J. Biol. Chem. 267 (1992) 5078^5084. [44] U.K. Laemmli, Nature 227 (1970) 680^685. [45] W. Wray, T. Boulikas, V. Wray, R. Hancock, Anal. Biochem. 118 (1982) 197^203. [46] G. Fairbanks, T.J. Steck, D.H.G. Wallach, Biochemistry 10 (1971) 2606^2612. [47] P.H.J. O'Farrell, J. Biol. Chem. 250 (1975) 4007^4021. [48] J.F. Cupo, G.P. Lidgard, W.F. Lichtman, Electrophoresis 11 (1990) 500^504. [49] H. Towbin, T. Staechelin, J. Gordon, Proc. Natl. Acad. Sci. USA 76 (1979) 4350^4354. [50] J.J. Leary, J.J. Brigati, D.C. Ward, Proc. Natl. Acad. Sci. USA 80 (1983) 4045^4049. [51] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265^275. [52] M. Watatani, T. Yoshida, K. Kuroda, S. Ieda, M. Yasutumi, Cancer 77 (1996) 1688^1693. [53] H.T. Lynch, T.C. Smyrk, P. Watson, S.J. Lanspa, J.F. Lynch, P.M. Lynch, J.R. Cavalieri, C.R. Boland, Gastroenterology 104 (1993) 1535^1549. [54] Y. Xing, C.V. Johnson, R.P. Dobner, J.B. Lawrence, Science 259 (1993) 1326^1330. [55] B. Chabot, S. Bisotto, M. Vincent, Nucleic Acids Res. 23 (1995) 3206^3213. [56] Ch.M. Clemson, J.A. McNeil, H.F. Willard, J.B. Lawrence, J. Cell Biol. 132 (1996) 259^275. [57] R.S. Izzo, C. Pellecchia, Scand. J. Gastroenterol. 33 (1998) 191^194. [58] R.S. Izzo, C. Pellecchia, Biochem. Mol. Biol. Int. 40 (1996) 521^526.
BBADIS 61933 19-5-00