TENASCIN-C SPLICE VARIANT EXPRESSION IN CULTURED HUMAN CHONDROCYTES AND CHONDROSARCOMA
CELLS
*Ghert, M A. (A-Orthopaedic Research and Education Foundation, Piedmont Foundation, Musculoskeletal Transplant Foundation); *Qi, W; *Erickson, H P.; **Block,
J A.; +*Scully, S P. (A-NIH (AR42863),OREF)
+*Duke University Medical Center, Division of Orthopaedic Surgery and Cell Biology, Durham, North Carolina. Duke University Medical Center, Box 3312, Durham,
NC 27710, (919)-681-6982, Fax: (919)-681-7161, [email protected]
Introduction : The ability of malignant cells to metastasize has been attributed to
the loss of cell-matrix adhesion [1,2]. Tenascin–C is a large hexameric
glycoprotein of the extracelleular matrix (ECM) that has been found to be
prominent in normal adult cartilage, embryogenesis, and epithelial and
mesenchymal tumors [3]. In addition, it has been shown to have anti-adhesive
properties, thus implicating the molecule in metastasis. However, recent
elucidation of several prominent splice variants of human tenascin-C and their
preferential distribution in developing and malignant tissue has introduced the
possibility of varying adhesive/anti-adhesive functions of tenascin-C based on its
specific splicing pattern.
The purpose of this study was to determine, using semi-quantitative reverse
transcription-polymerase chain reaction (RT-PCR) and Western blotting
techniques, the different in vitro tenascin-C splice variant expression and matrix
incorporation patterns of human chondroctyes (CS) and chondrosarcoma cells.
Methods: Cell culture and recovery: The phenotypically stable chondrosarcoma
cell line, JJ012, has been provided by Dr. Joel Block. Normal human
chondrocytes were harvested from operative specimens. Both cell types were
grown in a monolayer and when confluent, the cells were encapsulated in 1.2%
semi-solid alginate and maintained on 6-well plates at 2X104 cells/well. On days
3,5,7,9,15 and 21 of the bead culture, the media was aspirated from one well and
stored at -700C. The alginate beads from the well were dissociated by chelation and
the cells were freed from the buffer by centrifugation and both were stored at –
700C.Western Blotting: Cell lysate, media and alginate samples from each harvest
day were electropheresed on 5% SDS polyacrylamide gels under reducing
conditions with recombinant tenascin-C as a positive control. The proteins were
transferred to an immobilon membrane which was incubated in a polyclonal antitenascin antibody solution, rinsed, and incubated in a goat anti-rabbit IgG HRP
conjugate secondary antibody solution. The bound antibodies were exposed with
chemiluminesence (ECL) and the bands quantitated using densitometry and protein
concentration calculated based on the known concentration of the standards. RTPCR: Cells released from the alginate beads on the specific harvest days were
lysed and homogenized and total mRNA was extracted. First strand cDNA was
synthesized from the mRNA and primers for GAPDH, the variable splicing region
of tenascin (VS1) and the small splice variant (5/6) were designed using sequences
available in the GenBank database. PCR was performed and the products were run
on a 1% agarose/EtBr gel and photographed. Band density was evaluated using
densitometry and calculated as a ratio of GAPDH band density. Negative control
experiments excluded reverse transcriptase and cDNA.
All experiments were performed in triplicate. Statistical analysis was
performed using the students t-test comparison of means (Microsoft Excel). P<0.05
was considered statistically significant.
Results: Western Blot/Protein analysis:
A representative blot is shown in figure 1. The two major bands correspond to the
320 kDa and 220 kDa proteins. The JJ tumor cells produced small amounts of both
proteins and secreted both into the media. In contrast, the chondrocytes produced
significantly more of the 220 kDa protein, which was incorporated into the matrix
(alginate). Figure 2 demonstrates the significantly higher ratio of 220 kDa to 320
kDa produced and incorporated by the chondrocytes compared to the tumor cells.
Figure 1: Representative Western blot harvest day 9
 JJ tumor cells   chondrocytes 
M
A
C
M
A
C
320 kDa
220 kDa
M=media ,A=alginate, C=cell, JJ=tumor cells
Figure 2: Quantitative summary of protein data
RT-PCR results: The above results at the protein level were verified at the mRNA
expression level. On representative harvest days 9 and 15, mRNA expression for
the small splice variant was significantly higher in the chondrocytes than in the JJ
cells (Figure 3). In contrast, the expression for the large splice variant was the
same for both cells. This data was normalized to GAPDH for each sample and
semiquantitative results are shown in figure 4.
Figure 3: PCR products for small and large splice variants
 Small splice variant
Day 9
Day 15
CS
JJ
CS
JJ

Large splice variant 
Day 9
Day 15
CS
JJ
CS
JJ
Figure 4: Results of semiquantitave RT-PCR
Discussion: We have demonstrated at both the in vitro transcriptional and
translational levels that chondrosarcoma cells produce predominantly the 320 kDa
tenascin protein and secrete it into the media, whereas chondrocytes produce
predominantly the 220 kDa tenascin protein and incorporate it into the matrix. The
320 kDa variant has been associated with malignant cells, anti-adhesion and cell
movement, whereas the smaller variant has been associated with cell-matrix
adhesion [1,2]. Tenascin-C may therefore play a role in the metastatic cascasde
where the 220 kDa variant is required for the cell to anchor itself to the matrix and
prevent migration into the bloodstream. Futher eluciation of this mechanism may
lead to novel therapeutic approaches to chondrosarcoma.
References: [1] Kawakatsu et el.,JJCR 83:1073,1992. [2] Chiquet-Ehrisman et al.,
Canc.Bio
4:301,1993.
[3]
Vollmer
G,
CROH
25:187,
1997.
**Rush Presbyterian Medical Center, Chicago, IL.
Poster Session - Cartilage Cell Biology - Hall E
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California
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