These two isoforms differ in solubility, electrophoretic behavior, ... Sodium dodecyl sulfate -polyacrylamide gel ... guanidinium thiocyanate-phenol- ...

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TIlE Vol.



p. 233-241,








in Fibronectins



of Pathology,



of mRNA






: Demonstration


for publication


4 Human

Hepatoma-Associated Precursors

School of Medicine , Kurume, October

830 Japan

11, 1991

Summary : The synthesis and secretion of f ibronectin (FN) was observed in 4 human hepatocellular carcinoma (HCC) -derived cell lines using a serum-free conditioned medium. It was also demonstrated that FNs secreted from these cell lines (HCC-FN) were structurally altered compared to plasma FN produced by normal hepatocytes. Using limited proteolysis with cathepsin D followed by immunoblot analysis with a carboxy-terminal specific antibody , the existence of an extra domain (ED) segment in the carboxy-terminal region of HCC-FNs was confirmed. Expression of the additional nucleotide insert (ED sequence), which is reported to be absent in hepatocyte mRNA and is characteristic of cellular FN-mRNA, was confirmed in mRNA from all 4 HCC cell lines. It is now evident that HCC cells secrete different variants of FN compared with normal hepatocytes due to alternative splicing of mRNA precursors. This differential RNA processing seems to be one of the phenotypic alterations associated with the oncogenic transformation of hepatocytes . Key domain


f ibronectin










formed cells are altered by various mechanisms such as glycosylation, phosphorylation and alternative splicing of mRNA precursors (Ali and Hunter, 1981; Matsuura and Hakomori, 1985; Sekiguchi et al. 1985; Castellani et al. 1986). There are essentially two FN isof orms : one is "plasma" FN and the other is "cellular" FN (Sekiguchi et al. 1985, 1986). These two isoforms differ in solubility, electrophoretic behavior, certain biological functions, and the domain structure of the carboxy-terminal region. Recent studies involving cloning and sequencing of the FN gene and cDNA indicate that multiple mRNA species of FNs arise from a single gene by alternative splicing of the common mRNA precursors (Schwarzbauer et al. 1983; Kornblihtt et al. 1983, 1984a, 1984b, and 1985).


Fibronectins (FNs) are a group of high molecular weight adhesive glycoproteins present in plasma and extracellular matrices. They consist of several domains which have different biological functions and play an important role in cell-to-cell and cell-to-substratum adhesiveness, as well as in mediating various biological phenomena such as cell differentiation and maintenance of normal cell morphology (Pearlstein et al. 1980; Rouslahti et al. 1981b; Hynes and Yamada, 1982). A decrease or deletion of this protein associated with oncogenic transformation of cells has aroused a great deal of initial interest in the study of these proteins. Recently, many investigators have reported that FNs produced by trans233



Kornblihitt et al. (1984a), using the human breast cancer cell line Hs578T, have cloned a FN-cDNA containing an additional 270 nucleotide insert encoding for the extra domain (ED) segment which is not present in plasma FN produced by normal hepatocytes (Tamkun et al. 1983; Kornblihtt et al. 1984b). They also reported the existence of FN species with an ED segment in the HCC cell line Hep3B, that was similar to other cancer cell lines (Kornblihtt et al. 1984b). Today, there is great interest in differential RNA processing associated with the oncogenic transformation of hepa tocytes. In this report, an analysis of the structural alterations of FNs secreted from 4 human HCC-derived cell lines that are newly established is described, and ED-bearing FN species are demonstrated to be expressed in all these HCC cell lines.





Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 medium were obtained from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Fetal calf serum (FCS) was obtained from M. A. Bioproducts (Walkersville, MD, USA). Affinity purified rabbit IgG preparations against human plasma FN and monoclonal antibodies specific to the carboxy-terminal region of human plasma FN were purchased from DAKO (Copenhagen, Denmark) and Mallinckrodt Inc. (MO, USA), respectively. Gelatin-Sepharose 4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden) and restriction enzymes from Nippon Gene (Toyama, Japan). All other reagents were of analytical grade and obtained commercially.

KYN-1, KYN-2, KMCH-1) established in our laboratory (Murakami, 1984; Yano et al. 1986; Murakami et al. 1987; Yano et al. 1988) were grown in a humidified atmosphere of 5% C02 at 37°C. The cells were subcultured at confluence by standard trypsinization. The serum-free synthetic medium (SFM) used in the analysis of secreted proteins was RPMI 1640 supplemented with 3 x 10-8M sodium selenite and other trace elements according to the procedure of Nakabayashi et al. (1984). Spent culture media were harvested at 2-day intervals and stored at -35°C for subsequent analysis. Pooled media were centrifuged to remove cells and debris (2000 x g, 10 min.) and concentrated 100fold by an Amicon ultraf iltration concentrator (Amicon Corp., Lexington, MA, USA) using a diaf low membrane, YM10 (MW 103 cut-off). For isolation of the cellular RNA, the cells were maintained in DMEM supplemented with 10% heatinactivated FCS. Penicillin (100 u/ml) and streptomycin (100 µg/ml) were also added to each medium. The cell lines were morphologically well differentiated and retained several characteristic liver functions under the above culture conditions. Pooled plasma samples obtained from healthy individuals were used as normal controls for plasma FN. Duchterlony double -immunodiffusion: Double immunodif fusinn was performed with 1 °o agarose gel in phosphatebuffered saline (PBS) according to the standard method. Concentrated spent SFM were analyzed and the antigenicity of FNs, secreted from HCC cell lines (HCC-FNs), was compared to that of normal plasma using rabbit anti-human plasma FN antiserum. Purification HCC-FNs






cell lines


of FNs: and





pared by gelatin- Sepharose affinity chromatography, essentially with the method


of Engvall

and Rouslahti



Sodium dodecyl sulfate -polyacrylamide electrophoresis (SDS-PAGE) :


Protein samples were reduced by boiling for 5 min in SDS sample buffer (62.5 mM Tris-HCI, pH 6. 8, 5% 2-mercaptoethanol, 2 °o SDS, 10% glycerol, 0.01 % bromophenol blue) and separated on a 515°o gradient polyacrylamide slab gel containing 3°o stacking gel. The electrophoresis was carried out using the discontinuous buffer system described by Laemmli (1970). The gel was stained with Coomassie brilliant blue R250. Immunoblot


The proteins fractionated by SDSPAGE were electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA.) as described by Towbin et al. (1979). Following transfer, the membranes were incubated for 2 hr at room temperature in a solution of 20 mM Tris-HCI, (pH 7.4) and 150 mM NaCI (TBS) containing 3°0 bovine serum albumin (BSA) to saturate the nonspecific protein binding sites. The nitrocellulose membranes were then incubated with monospecific antibodies (polyclonal or monoclonal) to human plasma FN appropriately diluted in 1% BSA with TBS for 2 hr at room tempera ture. After being washed several times in TBS containing 0.05°o Tween 20, the specific antigen-antibody interactions were detected using the Vectastain ABC kit (Vector Laboratories Inc. Burlingame, CA.) and visualized by incubation with a substrate solution (0.05°0 4-chloro-1naphtol/0.05% 1120 2/16% methanol). Analysis of the domain structure carboxy-terminal region of FN: The terminal were

domain region compared

structures of by

of the

pFN limited


in the carboxy-

HCC-FNs proteolysis





using cathepsin D (Sigma . Chemical Co., St. Louis, M0.) followed by immunostaining of the released fragments with monoclonal antibodies specific to the carboxy-terminal region of human pFN, essentially by the procedure of Sekiguchi et al. (1985). Cathepsin D digestion of FN was performed with an enzyme/substrate ratio of 1:250, and the digests were analyzed on 8°o SDS-PAGE under reducing conditions. RNA preparation ization analysis:



blot hybrid-

Total cellular RNA was isolated from the confluent cell monolayers by an acid guanidinium thiocyanate-phenolchloroform extraction method (Chomczynski and Sacchi, 1987). Cellular RNA isolated from liver tissue, which was obtained from a non-cancerous region of surgically resected hepatoma tissue, was used as a control. Samples of 10 ,~g of total RNA were electrophoresed in formaldehyde agarose (1°0) gel according to the method of Maniatis et al. (1982). RNA was transferred to a nylon membrane (Hybond-N, Amersham) and fixed on the membrane by exposure to UV light. The membranes were prehybridized at 42°C for 4 hr in a solution of 5 x SSC (1 x SSC=0.15M NaCI and 0.015M sodium citrate), 50°0 (v/v) f ormamide, 0.1 °o Ficoll, 0.1 °o polyvinyl pyrrolidone, 0.1% BSA, 0.2% SDS, 50mM sodium phosphate and 100 ,ag/ml denatured salmon sperm DNA at pH 7.0. Hybridization was performed at 42°C for 40 hr in an identical solution containing 10% dextran sulfate and a denatured 32P-labeled DNA probe (5 x 106 cpm/ml). After hybridization, the membranes were washed at 55°C in solutions containing 0.1 °o SDS and decreasing concentrations of salt (2 x , 1 x , 0.5 x and 0.2 x 55C). They were then air-dried and exposed to Fuji RX-film with intensifying screens at -80°C.



Probe preparation:


The plasmid clone pFH111, which was isolated from the human breast cancer cell line Hs578T by Kornblihitt et al. (1984a) and which contains part of the FN-cDNA including the ED sequence, was obtained from the Japanese Cancer Research Resources Bank. This ED sequence is a 270 nucleotide insert which is located between the cell-binding (Cell) and carboxy-terminal heparin-binding (Hep-2) domains and can be included in or omitted from the FN-mRNA precursor depending on the pattern of RNA splicing. The EcoRI-EcoRI 1.6 kb insert of pFH111 was digested with the restriction enzyme Pst I, and a 180 by fragment containing the ED sequence of FN-cDNA was isolated by an electroelution method after separating the fragments in 3? 6'






2. Immunodiffusion FNs. Synthesis

cell normal SFM,


by in




demonstrated diffusion







Ouchterlony spent

The plasma





concentrated the



precipitation and




the lines

4 HCC for spent

to human

gel. This 180 by fragment was subcloned into the Pst I site of pUC19 vector (Bethesda Research Lab.). The plasmid clone, containing the 180 by fragment of the ED sequence, was purified after amplification and then radiolabeled with Ca-11P) dCTP by a multiprime DNA labeling system (Amersham) to a specific activity of 2-4X108 cpm/,erg DNA template, after the plasmid DNA was linearized by digestion with the restriction enzyme EcoRI.


1. Secretion of proteins cell lines.

from 4 human HCC

Equal amounts of proteins (25 ug) secreted in the SFM were analyzed on a 5-15°o gradient SDS-PAGE (Fig. 1). It was possible to identify many distinct bands of proteins with a prominent 67kdalton albumin band. The immunological identification of each secreted protein was reported previously (Fukuda et al. 1987). Thus, the 4 HCC cell lines were shown to retain the differentiated liver

Fig. 1. Comparison of profiles of proteins secreted by 4 human HCC cell lines with those of normal human plasma. Equal amounts of proteins (25 µg) were loaded on each gel lane and subjected to electrophoresis in a 5-15% gradient SDS-polyacrylamide slab gel. The gel was stained with Coomassie brilliant blue. The positions of the marker proteins of known molecular masses are indicated on the left. Lanes: 1=human plasma, 2=KIM-1, 3=KYN-1, 4=KYN-2, 5=KMCH-1







plasma FN, fused completely without distinct spurs (Fig-2). This suggests that there are no gross differences in antigenicity between plasma FN and HCC-FNs. 3. Molecular HCC-FNs.

Fig. reaction



of FNs of human

tured media. 1, 4=KYN-1,

precipitation plasma

Wells: 1=KMCH-1, 6=KYN-2, 2 and


cul -







The electrophoretic patterns of the subunits of HCC-FNs were compared to that of pFN by immunoblot analysis following 6% SDS-PAGE under reducing conditions (Fig. 3). Plasma FN showed double bands with a molecular weight of approximately 220 to 230 kd; whereas, each HCC-FN showed broader bands, with higher molecular weights than pFN.


Fig. 3. Immunoblot analysis of plasma FN and HCC-FNs using polyclonal (a) or monoclonal (b) antibodies. Plasma and spent SFM from HCC cell lines were fractionated by 6°6 SDS-PAGE under reducing conditions and electrophoretically transferred onto nitrocellulose membrane, as described in Materials and Methods. Lanes: 1= human plasma, 2-KIM-1, 3=KYN-1, 4=KYN-2, 5=KMCH-1


4. Comparison of the domain structure the carboxy- terminal region of FNs.



The variation in polypeptide structure between the FN isoforms are attributed to differences in the Hep-2 domain and/or its flanking regions. These differences give rise to characteristic fragments of the carboxy- terminal fibrin-binding (Fib2) and Hep-2 domains after mild cathepsin D digestion (Sekiguchi et al. 1985). Immunoblot analysis of the cathepsin D digest of pFN using carboxy-terminal region specific monoclonal antibodies revealed the fragments of Mr=70, 000 and 60, 000 daltons. In contrast, the corresponding fragments released from the HCC-FNs had much larger molecular weights than those from pFN (Fig. 4). These high-molecular-weight fragments of the carboxy-terminal region released by mild cathepsin D digestion suggest the existence of extra domain (s) around the Hep-2 and Fib-2 domains. 5. Expression of ED sequence in the FNmRNA species from cultured HCC cells. Total RNA from 4 HCC cell lines was analyzed by Northern blotting using a specific probe containing only the ED sequence. This probe was found to hybridize to one distinct band of the same size (about 8.0 kb) as mature FN-mRNA (Fig. 5). This indicates the existence of a FNmRNA species containing the ED sequence in all the cultured HCC cell lines. The ED sequence was not detectable in the cellular RNA isolated from control liver tissue.


The basic FN polypeptide has a structure with three different types of internal repeats (homology types 1, 11 and 111) and consists of several domain structures which have different binding activities

Fig. thepsin

4. Immunoblot D digests of

analysis plasma FN

FNs using carboxy-terminal oclonal antibodies. The lyzed under

on an reducing

ma FN, KYN-1


of the caand HCC-

specific digests were

8°o SDS-polyacrylamide conditions. Lanes: from




gel 1=plasfrom

(Pearlstein et al. 1980; Ruoslahti et al. 1981b; Hynes and Yamada, 1982). Recent analysis of the FN gene and cDNA indicates that the multiple mRNA species of FNs arise from a single gene, composed of more than 45 exons and introns, by alternative splicing of the initial RNA transcript (Schwarzbauer et al. 1983; Kornblihtt et al. 1983, 1984a, 1984b and 1985). It has previously been demonstrated that alternative splicing of the FN-mRNA precursor occurs mainly in at least two distinct regions termed IIICS (type III connecting segment) and ED (extra domain) (Kornblihtt et al. 1984b and







Fig. 5. Expression of ED sequence in HCC cells. Total RNAs were electrophoresed in a formaldehyde-agarose (loo) gel and transferred to a nylon membrane. The blots were hybridized with a probe containing the ED sequence. The cDNA map of the carboxy-terminal region of FN and the probe used in this study are also included. Lanes: 1=normal liver cells (negative control), 2=KIM-1, 3=KYN-1, 4=KYN-2, 5=KMCH-1

1985; Castellani et al. 1986). These two regions have been mapped to the Hep-2 domain and/or its flanking region (Sekiguchi et al. 1985). The FN protein variants corresponding to differentially processed mRNA have been identified as plasma-type and cellular-type FNs. Plasma FN is synthesized by hepatocytes, whose FN-mRNA is spliced so that it does not contain the ED sequence. The ED sequence has been identified as an additional 270 nucleotide insert located between the Cell and Hep-2 domains, and the existence of the ED sequence is believed to be characteristic of cellular FN (Kornblihtt et al. 1984b). However, IIICS is variably expressed in cellular and plasma FNs (Pande et al. 1987). In the present study, it was demonstrated that 4 different HCC cell lines secrete different FN variants into the culture media compared with normal hepatocytes. It was also shown that the me-

chanisms that mRNA precursor

regulate splicing of the are altered in HCC cells.

It is uncertain whether these alterations are due to the cell culture conditions or to the inherent characteristics of the malignant cells per se. However, several in vivo and in vitro investigations have indicated the existence of an extra domain in HCC-FNs. Kornblihitt et al. (1984b) also reported expression of the ED sequence in HCC-derived Hep3B cells. Matsuura et al. (1985) reported the existence of an unique FN domain in the carboxy-terminal region of FNs isolated from fetal tissue, placenta, amniotic fluid and various malignant tumors, including HCC tissue and cell lines. They proposed the term "oncofetal FN" for FNs containing an unique domain found in tumors and fetal tissue, but absent in normal adult tissue and plasma. Sekiguchi et al. (1986) reported that the FN isotype in tissue switches from the cellular (or fetal)-type to the plasma (or adult)-type



during ontogeriesis. Ruoslahti et al. (1981a) observed similarities in molecular weight and isoeiectric point between FNs from human germ cell tumors and amniotic fluid, which were also distinctly different from plasma FN. They suggested that such FNs may provide onco developmental markers. In the current investigation, the ED segment was expressed in all of the 4 different HCC cell lines, and it is now evident that expression of ED-bearing FN species is one of the distinct phenotypic alterations associated with the oncogenic transformation of hepatocytes. Although little is known about the components involved in RNA splicing and how it is regulated, the FN gene in HCC cell lines may serve as a model for the study of oncogenic modulation of RNA splicing. Furthermore, it will be of great interest to evaluate the ED-bearing FN species as a tumor marker for HCC. Acknowledgments thank Prof. M.Kojiro The author would members in the First for helpful assistance.

: The author wishes to for supporting this work. also like to thank other Department of Pathology


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