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Proteoglycans from the substratum adhesion sites of MSV-transformed BALB/c 3T3 cells
Abstract:
Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 M guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material--high-density GA1 which contained some HA but principally CS and HS; intermediate-density GA2 which contained only HA; and low-density GA3 which contained some HA and principally glycoprotein. R-SAM gradients contained no GA2 but a sizable amount of "low-density" HA in GA3. When centrifuged under dissociative conditions, most of GA1 and all of GA2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have "matured," indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.
Insights
Kirsten murine sarcoma virus-transformed cells (KiMSV) alter their adhesion sites, increasing hyaluronate (HA) and chondroitin sulfate (CS) content. These changes in proteoglycans (PGs) influence cell detachment and extracellular matrix interactions.
Area of Science:
- Biochemistry
- Cell Biology
- Extracellular Matrix Research
Background:
- Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) exhibit high tumorigenicity and metastatic potential.
- These transformed cells display altered adhesion properties, detaching readily from substratum.
- Adhesion sites left by detached cells contain specific extracellular matrix components.
Purpose of the Study:
- To characterize the proteoglycan (PG) composition of adhesion sites generated by KiMSV cells.
- To compare the PGs in adhesion sites of transformed KiMSV cells with those of parental Balb/c 3T3 cells.
- To investigate the role of specific glycosaminoglycans in cell adhesion and detachment.
Main Methods:
- Isolation and biochemical analysis of substratum-attached material (SAM) from KiMSV and Balb/c 3T3 cells.
- Extraction of proteoglycans using 4 M guanidine hydrochloride.
- Characterization of proteoglycans using Sepharose CL2B chromatography and isopycnic density gradient centrifugation.
- Analysis of glycosaminoglycan composition, including hyaluronate (HA), chondroitin sulfate (CS), and heparan sulfate (HS).
Main Results:
- KiMSV cell adhesion sites (L-SAM and R-SAM) are enriched in HA and CS compared to parental 3T3 cells, which show higher levels of heparan sulfate (HS) in R-SAM.
- KiMSV proteoglycans exhibit reduced aggregation into high-molecular-weight complexes but form intermediate-size complexes not observed in 3T3 cells.
- Density gradient analysis revealed distinct PG populations in KiMSV L-SAM (GA1, GA2, GA3) and R-SAM, with significant amounts of HA in GA2 and GA3.
- Dissociative centrifugation showed differential behavior of PGs from L-SAM and R-SAM, indicating distinct structural properties.
Conclusions:
- KiMSV cells generate adhesion sites with a distinct proteoglycan composition compared to parental 3T3 cells.
- The altered PG profile, particularly increased HA, supports its role in facilitating cell detachment from the extracellular matrix.
- Newly formed HS-PGs in KiMSV R-SAM differ significantly from matured sites, suggesting ongoing metabolic activity and remodeling within adhesion sites of transformed cells.