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[Cancer Research 60, 1117-1120, February 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Nuclear Matrix of Calreticulin in Hepatocellular Carcinoma1

Ghil-Suk Yoon, Hojung Lee, Yusun Jung, Eunsil Yu, Hee-Bom Moon, Kyuyoung Song and Inchul Lee2

Departments of Pathology [G-S. Y., H. L., Y. J., E. Y., I. L.], Internal Medicine [H-B. M.], and Biochemistry [K. S.], University of Ulsan College of Medicine and Asan Institute for Life Sciences, Seoul, Korea


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear matrix protein profiles of malignant cells vary from their normal counterparts. By two-dimensional gel electrophoresis, we analyzed nuclear matrix proteins in 11 hepatocellular carcinomas and compared them with corresponding non-neoplastic liver tissue. Although the compositions were mostly similar, several peptides were noted predominantly in the former. The most prominent one was an acidic protein of apparent Mr 62,000, which was identified to be calreticulin upon NH2-terminal amino acid sequencing. By immunoblotting, calreticulin was confirmed to be present abundantly in the nuclear matrix fraction of carcinomas but not in that of the nonmalignant liver tissue. Interestingly, the total content of calreticulin was similar between them. By immunofluorescence microscopy, evident nuclear immunostaining was detected in carcinomas. Calreticulin was also found to be in the nuclear matrices of various carcinoma cell lines. We conclude that calreticulin is a component of the nuclear matrix. The formation and/or expansion of the calreticulin-nuclear matrix may be related to the activated cell growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear matrix is a nonchromatin structure that resists extraction with detergents, high ionic strength salt, and DNases (1) . It consists of nuclear matrix proteins and ribonucleoprotein complexes and has been implicated in the regulation of DNA replication, transcription, and RNA processing (reviewed in Ref. 2 ).

Depending on cell types, nuclear matrix proteins may vary considerably. Although the majority are common in most cell types, certain proteins have been reported to be specific for particular cell types (3, 4, 5, 6) . Furthermore, it was reported that transformed cells with high metastatic potential displayed altered nuclear matrix protein profiles (7) . Thus, it was suggested that a group of nuclear matrix proteins was exclusively expressed or "recruited" in association with malignant transformation or other related cellular changes.

Using two-dimensional gel electrophoresis, we analyzed the nuclear matrix protein profiles of human HCCs3 and compared them with corresponding non-neoplastic liver tissue. Several proteins were noted predominantly in carcinomas: unexpectedly, one of them was identified as calreticulin. Here we report that a fraction of cellular calreticulin participates in the formation of the nuclear matrix in HCCs and other carcinoma cell lines.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples and Cells.
Fresh tumor and non-neoplastic liver samples were obtained from 11 partial hepatectomy specimens. Patients were from 37 to 72 years of age; eight had hepatitis B virus infection. Tumors varied from 3 to 12 cm in diameter and displayed typical pathological features of well or moderately differentiated HCCs. Adjacent liver tissues were also sampled: they displayed either cirrhosis or chronic hepatitis. In addition, three normal liver samples were obtained from specimens resected for hepatolithiasis. Samples were immediately frozen in liquid nitrogen and kept at -70°C until they were used.

Hep3B, HepG2, HeLa, and EJ cells were grown in DMEM with 10% FCS. Cultures were incubated at 37°C and 4% CO2 in 75 cm2 tissue culture flasks. Cells were harvested when they reached 50% confluence.

Preparation of Nuclear Matrix Proteins.
Nuclear matrix proteins were prepared essentially according to Fey and Penman (3) . Briefly, the sample was minced in PBS and washed with PBS three times. It was resuspended and homogenized in buffer A [10 mM PIPES (pH 7.0), 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1.2 mM phenylmethylsulfonyl fluoride, 2 mM vanadylribonucleoside, and 0.5% Triton X-100]. The homogenate was filtered through four layers of cheesecloth and was spun down at 1000 x g for 10 min at 4°C. The pellet was resuspended in buffer B (100 µg/ml DNase I added to buffer A) for 45 min at 25°C and centrifuged at 1000 x g for 10 min at 4°C. The pellet was resuspended in buffer C (250 mM ammonium sulfate added to buffer A), stirred in cold room for 10 min, and centrifuged at 1000 x g for 10 min at 4°C. The pellet was resuspended in buffer A. While it was stirred at 4°C, 4 M stock solution of NaCl was added slowly until the final concentration of NaCl reached 2 M. After 10 min of incubation, it was centrifuged at 1000 x g for 10 min. The pellet, which represented a crude nuclear matrix fraction containing intermediate filament proteins as well, was applied to two-dimensional gel electrophoresis.

Nuclear matrix proteins of cultured cells were prepared similarly. After washing with PBS, harvested cells were resuspended in buffer A and homogenized in a loose-fit glass-to-glass homogenizer. After centrifugation, the supernatant was collected as "total soluble cytoplasmic protein fraction." The pellet, a "total crude nuclear protein fraction," underwent further extraction procedures as above. To see the effect of Ca2+ on the extraction, the procedures were repeated with the same buffers but without EGTA.

Two-Dimensional Gel Electrophoresis.
Two-dimensional gel electrophoresis was carried out essentially according to O’Farrell (8) . Nuclear matrix fractions were applied to a lysis buffer [9.8 M urea, 2% (w/v) NP40, 100 mM DTT, and 2% Ampholine (pH 7–9; Pharmacia Biotech, Piscataway, NJ)] and were applied to isoelectric focusing. The tube gels were extracted and applied to 10% SDS-PAGE. Slab gels were fixed and stained with Coomassie Blue. For normalization of the loaded proteins, the amount of cytokeratin in the crude nuclear matrix fraction was measured by densitometry and used as a standard.

NH2-Terminal Sequencing.
For NH2-terminal amino acid sequencing, peptides were transferred to Immobilon membranes (Hoefer Scientific, San Francisco, CA). After staining with Coomassie Blue, the candidate peptide was cut out. The amino acid sequencing by Edman degradation method was carried out at the Korean Basic Science Institute (Daeduk, Korea).

Amino Acid Sequence Analysis.
The nonredundant protein sequence database at the National Center for Biotechnology Information (NIH) was searched for homology using the BLASTP program. For the prediction of coiled-coil structure, the amino acid sequence of calreticulin was analyzed using the Network Protein Sequence analysis supplied by Pôle Bio-Informatique Lyonnais (Lyon, France; Ref. 9 ).

Immunoblotting.
Proteins were separated by SDS-PAGE and then transferred to nitrocellulose membranes. For immunoblotting, a rabbit anticalre-ticulin antibody (StressGen, Victoria, British Columbia, Canada) was applied in 1:500 dilution. Then membranes were applied to either the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reaction system (Boehringer-Mannheim, Mannheim, Germany) or the enhanced chemiluminescence system (Amersham, Buckinghamshire, England).

Immunofluorescence Microscopy.
Four-µm-thick frozen sections were made from every malignant and normal tissue. They were fixed in cold acetone at -20°C for 10 min and then anticalreticulin antibody was applied at 1:500 dilution. For a negative control, a nonimmune rabbit serum was applied. For simultaneous DNA staining, 4',6-diamino-2-phenylindole (Sigma, St. Louis, MO) was applied before mounting.

Cultured cells were similarly studied for calreticulin expression. Cells were grown on coverslips to 50% confluence and were washed in PBS. They were fixed in cold methanol at -20°C for 10 min and then were processed similarly.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Calreticulin in the Nuclear Matrix Fraction.
Upon two-dimensional gel electrophoresis, similar numbers of peptides were identified in the nuclear matrix preparations of malignant and normal liver tissues, respectively (Fig. 1, A and B)Citation . The electropherogram patterns were quite similar. Individual variations among the samples were insignificant.



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  		Fig. 1. Two-dimensional gel electrophoresis of nuclear matrix proteins. Nuclear matrix proteins from a HCC (A) and non-neoplastic liver tissue from the same patient (B) were analyzed. The amount of cytokeratin (arrow) was measured by densitometry and used as a standard for sample loading. Whereas most peptides appear to match, several peptides were noted only in the tumor (A). The most notable one with apparent Mr 62,000 and pI 4.0 turned out to be calreticulin (empty arrow), which was not detected in normal tissue (B). Gels were prepared by isoelectric focusing and Coomassie Blue staining. Left, molecular weight in thousands.

 
In the Coomassie Blue-stained gels, several proteins were observed only or predominantly in carcinomas. Among the proteins, the most prominent one was with apparent Mr 62,000 and pI 4.0 (Fig. 1A)Citation . It was readily detected in the nuclear matrix fractions of 10 of 11 carcinomas. The peptide was submitted for NH2-terminal amino acid sequencing. From the eluted peptide, 14 amino acids were sequenced: VYFKEQFLDGDGWT. According to the sequence homology search, they were identical to residues 4–18 of human calreticulin (10) . The pI and apparent molecular weight on SDS-PAGE were compatible with those of calreticulin.

To confirm the presence of calreticulin, the nuclear matrix fractions of carcinomas and normal liver tissues were immunoblotted with an anticalreticulin antibody. Calreticulin was confirmed to be one of the major components of the nuclear matrix proteins in carcinomas; however, it was minimal or barely detectable in nuclear matrix fractions of normal liver (Fig. 2)Citation .



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  		Fig. 2. Calreticulin in the nuclear matrix fraction of HCC. A, 10% SDS-PAGE of nuclear matrix protein fractions of normal liver tissue and tumor. The same amount of each protein (12 µg) is loaded. Calreticulin is noted as a major protein (arrow). The band of similar molecular weight in normal liver represents apparently unrelated protein(s). B, immunoblot of the same samples as used in A. Calreticulin is readily identified in the nuclear matrix of tumor (arrow), whereas it is barely visible in the nuclear matrix of normal liver. Left, molecular weight in thousands.

 
Because calreticulin was a major calcium-binding protein, it was suspected that the concentrations of calcium ions in the buffers might have affected the solubility or the extraction. When the extraction procedures were repeated using the same buffers but without EGTA, no recognizable difference was noted; calreticulin was abundant in the nuclear matrices of carcinomas but not in those of normal liver tissue (data not shown).

The detection of calreticulin in the nuclear matrix fraction might have represented an "incomplete extraction" of calreticulin if it was abundant in the carcinomas particularly. To analyze such a possibility, the total calreticulin contents in the samples were compared. Sample buffers containing the same amount of carcinoma and normal liver tissue samples were loaded in the SDS-PAGE and transferred to a nitrocellulose paper. Upon immunostaining with an anticalreticulin antibody, the relative intensity of calreticulin immunoreaction was not increased in carcinomas (Fig. 3)Citation . On the contrary, it appeared to be slightly less in carcinomas than in normal tissues; however, the difference was not considerable.



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  		Fig. 3. Calreticulin in the whole cell proteins of normal and neoplastic liver tissue. A, 10% SDS-PAGE of whole cell proteins of three non-neoplastic liver tissues (Lanes 1–3) and HCCs (Lanes 4–6). The same amount of each protein (15 µg) is loaded in every lane. B, immunoblot of the same samples as used in A. Calreticulin appear to be included slightly more in Lanes 1–3 than in Lanes 4–6; however, the difference is not considerable. Left, molecular weight in thousands.

 
Immunolocalization of Calreticulin in the Nuclei of Carcinomas.
To confirm the presence of calreticulin in the nucleus of carcinoma cells, we examined intracellular localization patterns of calreticulin by immunofluorescence microscopy. In the noncarcinomatous tissues, calreticulin was detected extensively in the cytoplasm of hepatocytes and Kupffer cells (Fig. 4A)Citation ; the latter tended to be more intensely immunostained than the former. No convincing nuclear staining was noted except for occasional small dot-like immunoreactivity.



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  		Fig. 4. Immunofluorescence microscopy for calre-ticulin. A, HCC displaying diffuse cytoplasmic and patch nuclear staining (arrows). The nuclear patches vary in number and size considerably. B, normal liver tissue from the same patient as in A. Hepatocytes are diffusely stained in the cytoplasm. More strongly stained cells correspond to Kupffer cells. No evident nuclear staining is noted in any cell type. C, Hep3B cells. Some cells display evident nuclear as well as cytoplasmic staining. D, double staining of the same cells as in C for DNA (4',6-diamino-2-phenylindole staining). A–D, x300.

 
The carcinoma cells also displayed diffuse and intense cytoplasmic staining. They also frequently displayed evident nuclear staining, which consisted of irregular patches of various sizes ranging from 1 to 5 µm (Fig. 4B)Citation . Typically, several patches were present in a nucleus; the distribution and shape were distinct from those of nucleoli. Diffuse and weak nuclear staining was noted frequently as well.

Nuclear immunostaining for calreticulin was noted in Hep3B and HeLa cells as well (Fig. 4, C and D)Citation . The nuclear staining was either patchy or diffuse. Compared with those cells, the nuclear staining was rather weak and ambiguous in EJ and HepG2 cells.

Calreticulin in the Nuclear Matrix of Various Cells.
Nuclear matrix proteins of HepG2, HeLa, and EJ cells were immunoblotted with an anti-calreticulin antibody. Calreticulin was particularly abundant in the nuclear matrix of HeLa, whereas the amounts were much smaller in EJ and HepG2 (Fig. 5A)Citation . However, it was difficult to compare the amounts directly because the pellets of nuclear matrix of HeLa tended to be larger than those of EJ and HepG2 after the extraction process. Interestingly, the calreticulin immunoreactivity was frequently noted as two separate bands (which apparently were Mr 62,000 and 64,000 in our gel system), particularly in EJ and HepG2. It did not appear to represent a proteolytic degradation in the procedures because it was noted in total tissue or cell preparations quite often (data not shown).



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  		Fig. 5. Immunoblotting analysis of calreticulin. A, nuclear matrix proteins of EJ (Lane 1), HeLa (Lane 2), and HepG2 (Lane3). Nuclear matrix fractions from 6 x 106, 1 x 106, and 6 x 106 cells are loaded, respectively. Note two positive bands with Mr of approximately 62,000 and 64,000 in the samples, particularly in EJ (arrows). B, total cytoplasmic protein fraction (Lane 1), total crude nuclear protein fraction (Lane 2), and nuclear matrix fraction (Lane 3) of HeLa cells. Each lane represents fractions from 4 x 103, 2 x 106, and 2 x 106 cells, respectively. Mr markers represent 250,000, 98,000, 64,000, 50,000, 36,000, and 30,000, respectively. Blots were visualized by chemiluminescence method.

 
By immunoblotting, the relative amounts of calreticulin were compared in HeLa cell fractions of "total soluble cytoplasmic protein," "total crude nuclear protein," and "nuclear matrix," respectively. It was estimated that calreticulin in the nuclear matrix fraction consisted of about 0.1 and 10% of the total calreticulin in the cytoplasm and nucleus, respectively (Fig. 5B)Citation .

Prediction of Coiled Coil in Calreticulin.
When the amino acid sequence of calreticulin was analyzed with the window at 28 amino acids (9) , a segment (amino acid residues 350–408) at the COOH terminus predicted a virtual coiled coil (Fig. 6)Citation . It encompassed about 14% of the entire amino acid sequence of calreticulin.



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  		Fig. 6. Prediction of coiled coil of calreticulin. The Y axis denotes the probability, and the X axis denotes amino acid numbers from the NH2 terminus. The COOH terminus from amino acids 350 to 408 predicts a virtual coiled coil.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It was an unexpected finding that calreticulin was in the nuclear matrix of malignant cells because it was known to be mostly in the endoplasmic reticulum (reviewed in Ref. 10 ). Calreticulin is a ubiquitous calcium-binding protein, which some have suggested is an autoantigen (11) . It has important regulatory functions such as modulation of steroid hormone receptors (12 , 13) , integrin functions (12 , 13) , and retinoid acid receptors (14) . It has been shown to regulate vinculin expression (15) and integrin-mediated calcium signaling (16) .

Calreticulin is a highly acidic protein that moves aberrantly at about Mr 60,000–65,000 on SDS-PAGE, although the deduced Mr from the amino acids is 46,000 (10 , 17 , 18) . Because it is an abundant protein with unusual physicochemical characteristics, the possibility of an inadequate extraction or an artifact in the procedure should be considered. However, such a possibility is very unlikely. Under the same extraction procedures, calreticulin was consistently found in the nuclear matrix fractions of carcinomas, whereas in the nuclear matrix fractions of normal liver tissue (which had the same amount or even more total calreticulin) it was minimally present. Furthermore, by immunofluorescence microscopy, it was evidently present in the nuclei of HCCs, whereas normal hepatocytes did not display convincing nuclear staining. Thus, it was concluded that calreticulin was indeed present in the nuclei and that a fraction of nuclear calreticulin was a component of the nuclear matrices of HCCs.

The prediction of a segment of virtual coiled coil also supports the notion that calreticulin might function as a structural protein in the nucleus. Although the predicted coiled coil is not as long as those of known filamentous proteins such as intermediate filaments or lamins, calreticulin may interact with other nuclear proteins with coiled-coil domains, depending on the microenvironmental situation. Because only a small fraction of nuclear calreticulin is detected in the nuclear matrix fraction, such a possibility appears to be high.

The intracellular localization of calreticulin has been a controversial issue. In addition to being a calcium reservoir in the endoplasmic reticulum, calreticulin has important gene-regulating functions. It has been suggested that the regulatory functions of calreticulin are manifested by direct binding to a common amino acid sequence motif KXFFKR (where X is either G, A, or V) at the DNA binding domain of all steroid receptors or to a homologous motif KXGFFKR of the cytoplasmic domains of all integrin {alpha} subunits (12, 13, 14) . If it regulated the hormonal receptor functions by blocking their binding sites to DNA directly, it would be expected to be at the very site of DNA binding, i.e., the nucleus.

Calreticulin has a hydrophobic leader sequence at the NH2 terminus and the KDEL motif at the COOH terminus, which are characteristic for proteins retained in the endoplasmic reticulum (19) . However, a putative nuclear localization signal, -PPKKIKPDP-, is present at residues 187–195 as well. It would be compatible with the notion of direct interaction of calreticulin with hormonal receptors in the nucleus. On the contrary, it was shown that only the "endoplasmic reticulum form" of calreticulin had regulatory functions of hormonal receptor-mediated gene expression (20) .

Results of immunofluorescence studies have been controversial as well. In early studies, positive nuclear immunostaining was reported (21 , 22) . However, in a subsequent study using isolated rat hepatocytes, no nuclear staining was observed (20) . Indeed it was not incompatible with our observation because convincing nuclear staining was observed only in the malignant cells and not in normal hepatocytes. Thus, it could be concluded that calreticulin was able to get into the nucleus under certain cellular conditions, including malignant transformation. The "intranuclear translocation" of calreticulin appeared to be a strictly regulated biological phenomenon.

The nuclear matrix is a highly dynamic structure that may be formed or modified readily according to the microenvironment. Recently, it has been reported that a fraction of Ku, a DNA-binding protein, was present in the nuclear matrix as well as in the "soluble" fraction (23) . Thus, it is not unprecedented that a fraction of a "soluble" protein like calreticulin may participate in the formation of the nuclear matrix, depending on the biological conditions.

The nuclear matrix of calreticulin may not be restricted to hepatic tissue or specific for a malignancy. Calreticulin may still be detectable, although in a much lower amount, in the nuclear matrix fractions of normal tissues. If calreticulin were indeed in the nuclear matrix normally, such a phenomenon appears to be augmented considerably during the malignant transformation. Otherwise, the presence of calreticulin in the nuclear matrix may be related to the "activated cell growth" in general. In this context, it is of note that the expression of calreticulin is increased in T lymphocytes as they are activated (24) .


   ACKNOWLEDGMENTS
 
We thank Yeonhee Choi, Mijoo Rhru, Seongsoo Kim, and Sujung Kang for technical help.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by funding for basic medical sciences from the Ministry of Education (1997-146) and by Grants 1997-7 and 1998-7 from the Asan Institute for Life Sciences. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Gu, Seoul 138-736, Korea. Phone: 82-2-2224-4551; Fax: 82-2-472-7898; E-mail: iclee{at}www.amc.seoul.kr Back

3 The abbreviations used are: HCC, hepatocellular carcinoma; pI, isoelectric point. Back

Received 4/ 5/99. Accepted 12/14/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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G. Brunagel, B. N. Vietmeier, A. J. Bauer, R. E. Schoen, and R. H. Getzenberg
Identification of Nuclear Matrix Protein Alterations Associated with Human Colon Cancer
Cancer Res., April 1, 2002; 62(8): 2437 - 2442.
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F. Le Naour, F. Brichory, D. E. Misek, C. Brechot, S. M. Hanash, and L. Beretta
A Distinct Repertoire of Autoantibodies in Hepatocellular Carcinoma Identified by Proteomic Analysis
Mol. Cell. Proteomics, March 1, 2002; 1(3): 197 - 203.
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