Wen-Ning Qi

Interstitial collagenase gene expression correlates with in vitro invasion in human chondrosarcoma

Matrix metalloproteinases contribute to the processes of local invasion and metastasis by providing cells with the ability to traverse tissue boundaries. The levels of gene expression were quantitated for matrix metalloproteinases-1 and tissue inhibitors of metalloproteinases-1 in human chondrosarcoma cell lines, and the results were correlated with cell differentiation, collagenase activity, and in vitro invasion. Three well characterized human cell lines were used in this study, with the level of chondrocytic differentiation confirmed to be JJ012, FS090, and 105KC in increasing order on the basis of aggrecan and collagen gene expression. The matrix metalloproteinases-1/tissue inhibitors of metalloproteinases-1 ratio correlated with the level of differentiation in an inverse fashion. Collagenase activity paralleled matrix metalloproteinases-1/tissue inhibitors of metalloproteinases-1 gene expression and was associated with a more invasive phenotype in an in vitro assay. In this report, matrix metalloproteinase-1 and tissue inhibitors of metalloproteinases-1 expression in human chondrosarcoma tumor cell lines were quantitated, and it was shown that interstitial collagenase gene expression correlates inversely with chondrocytic differentiation. Differences in collagenase activity and in vitro invasion correlate inversely with the level of differentiation. These findings are consistent with the hypothesis that collagenase activity is associated with a poorer prognosis in chondrosarcoma by facilitating cell egress from the tumor matrix.

siRNA mediated inhibition of MMP-1 reduces invasive potential of a human chondrosarcoma cell line

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP‐1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP‐1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP‐1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP‐1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP‐1 gene expression and protein synthesis was demonstrated via RT‐PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP‐1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP‐1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP‐1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.

Inhibition of MMP-1 expression by antisense RNA decreases invasiveness of human chondrosarcoma.

We previously reported that an elevated level of matrix metalloproteinase‐1 (MMP‐1) gene expression in patients with chondrosarcoma has a strong statistical correlation with recurrence and in vitro invasion. In the present study, we used an antisense RNA strategy for MMP‐1 inhibition to determine if this would affect the invasive characteristics of the cells. We transfected a human chondrosarcoma cell line with a retroviral plasmid expressing a 770 bp genomic fragment of the human MMP‐1 gene in the sense or antisense orientation. The results show that cells transfected with the MMP‐1 antisense fragment had a significant decrease in both MMP‐1 protein and enzyme activity (p < 0.05) as compared to cells transfected with an empty plasmid or the parental cells. Cells transfected with the MMP‐1 antisense fragment demonstrated a significant decrease in their ability to invade the collagen I barrier (p < 0.05). The gene expression for MMP‐8 and MMP‐13 were unaffected in cells transfected with the MMP‐1 antisense fragment, MMP‐1 sense fragment, or empty plasmid. These results support the hypothesis that MMP‐1 facilitates tumor cell egress from chondrosarcoma tissue and demonstrate the potential of MMP‐1 as a promising target for a novel biologic therapy in chondrosarcoma.

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection.

The Clinical Significance of Tenascin-C Splice Variant Expression in Chondrosarcoma

Objectives: Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix that is prominently expressed in malignant tumors. The purpose of this study was: (1) to determine the in vitro TNC splicing pattern in cultured human chondrocytes and chondrosarcoma cells, (2) to determine the in vivo TNC splicing pattern in clinical chondrosarcoma specimens, and (3) to perform survival analysis based on the TNC splicing pattern of the tumor specimens. Methods: Human articular chondrocytes and chondrosarcoma cells (cell line JJ012) were grown in a three-dimensional alginate bead system and harvested at two time points. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the in vitro TNC splicing pattern for the two cell types. Clinical chondrosarcoma specimens were obtained intra-operatively and underwent RT-PCR to determine the in vivo TNC splicing pattern. Specific immunohistochemical staining for the large TNC splice variant was performed on the clinical specimens. Survival analysis was used to determine the association between the specific TNC splicing pattern and survival. Results: The in vitro mRNA expression pattern of TNC in normal human articular chondrocytes was characterized by a high ratio of the small to the large splice variant (TNCsmall:TNClarge), whereas the in vitro mRNA expression pattern for cultured chondrosarcoma cells was characterized by a low TNCsmall:TNClarge ratio. Clinical chondrosarcoma specimens with a lower TNCsmall:TNClarge ratio showed a trend towards decreased survival. The TNC splicing pattern of these specimens was verified through specific immunohistochemical staining for the large TNC isoform. Conclusions: The specific TNC splicing pattern may have clinical significance in chondrosarcoma. TNC expression may therefore play a future role in objecitve tumor grading and novel therapeutic approaches to this malignancy.

Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes

The articular cartilage extracellular matrix (ECM) interfaces with chondrocytes and influences many biological processes important to cartilage homeostasis and repair. The alginate bead culture system can be viewed as a model of cartilage repair in which the chondrocyte attempts to recreate the pericellular matrix while maintaining a differentiated phenotype. The purpose of this study was to evaluate the alteration in epitopes of proteoglycan and tenascin synthesized by chondrocytes in the presence of exogenous extracellular type II collagen. We evaluated the effects on four biomarkers associated with the creation of the denovo matrix using ELISA and immunohistochemistry: keratan sulfate epitope (5D4), 3B3(−) neoepitope of chondroitin‐6‐ sulfate, 3B3(+) chondroitinase‐generatedepitope of chondroitin‐6‐sulfate, and tenascin‐C expression. TGF‐β1 stimulated the production of 3B3(+), 5D4, and tenascin‐C in a dose‐dependent manner and decreased 3B3(−) levels. Following the addition of exogenous type II collagen, 3B3(−) increased and tenascin‐C decreased but did not change the direction of TGF‐β1 effects. In contrast, 5D4 expression decreased in the presence of collagen II as TGF‐β1 increased to 10 ng/ml. Interestingly, the amount of 3B3(+) epitope was not affected by the incorporation of type II collagen. Immunohistochemistry found there was no significant difference in distribution of these biomarkers in the presence and absence of extracellular type II collagen incorporation. These results elucidate the subtle biochemical differences in ECM synthesized by chondrocytes in the presence of type II collagen and further characterize the role played by ECM in the TGF‐β1 regulation of the articular cartilage physiology.

Tenascin-C expression and distribution in cultured human chondrocytes and chondrosarcoma cells

Tenascin‐C (TNC) is an oligomeric glycoprotein of the extracellular matrix with several distinct isoforms variably expressed during embryogenesis, tumorogenesis, angiogenesis and wound healing. In the normal human adult, TNC is found in large concentrations in articular cartilage, suggesting tissue‐specific function. The purpose of this study was to determine the specific in vitro TNC splicing patterns of articular chondrocytes and a human chondrosarcoma cell line. Cells were cultured in a three‐dimensional bead system and TNC splice variant expression and distribution were examined with the use of Western blotting techniques, semi‐quantitative reverse‐transcription polymerase chain reaction and immunohistochemistry. At both the transcriptional and post‐translational levels, the chondrocytes were found to express significantly higher levels of the smaller 220 kDa isoform (P < 0.01), which was predominantly incorporated into the matrix. The splicing pattern of the malignant cells was characterized by a higher proportion of the larger 320 kDa isoform which was extruded into the media. In vivo studies are necessary to verify the expression of the large TNC isoform in chondrosarcoma and the production and integration of the smaller isoform in normal chondroid matrix. In addition, elucidation of the biologic functions of the two major TNC isoforms may lead to the development of novel diagnostic and therapeutic approaches to chondrosarcoma.

Gelatinase and inhibitor expression in soft tissue sarcomas: lack of correlation with distant metastasis.

The predominant mode of death for most patients with soft tissue sarcomas (STS) remains distant metastasis (DM). Current clinical predictors of DM are unreliable. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) correlate with biologic aggression in other tumors. The gene expression of the gelatinase, MMP-2 and MMP-9, and their respective inhibitors, TIMP-1 and TIMP-2, in STS was evaluated. Twelve fresh-frozen surgical specimens from patients with large (>5 cm) STS were analyzed. Six patients developed DM while 6 survived disease-free (DFS) at a minimum follow-up of 13 months. Following mRNA isolation, reverse transcription-polymerase chain reaction was performed using primers for MMP-2, MMP-9, TIMP-1, and TIMP-2. Gene expression was determined by band densitometry. Ratios of MMP-9/TIMP-1 and MMP-2/ TIMP-2 gene expression as well as MMP-2 protein activation ratio (active/inactive enzyme determined by gelatin zymography) were analyzed for correlation with DM and DFS. MMP-2 gene was expressed in 12 specimens, while MMP-9 was detectable in 9. Relative levels of MMP-2 and MMP-9, MMP2/TIMP-2 ratio, and MMP-9/TIMP-1 ratio were not significantly correlated with DM. Poor DFS was significantly correlated with high MMP-9/TIMP-1 ratio (p = 0.02). Active MMP-2 protein was detected in 12 specimens, while active MMP-9 protein was detected in 2. No association was found between MMP-2 protein activation ratio and DM or DFS. While MMP-2 gene expression and protein activity occurred in these 12 specimens, gelatinase/inhibitor ratios (for both MMP-2 and MMP-9) appear to be poor predictors of DM in STS.