Article Figures & Data
Figures
- Fig. 1.
Expression of BEHAB/brevican in rodent and human brain tumors. A, Western blots of invasive rodent brain tumors established from CNS-1 cells show full-length 140 kDa BEHAB/brevican, as well as 90 and 50 kDa proteolytic fragments. Full-length and cleaved 90 kDa bands are visualized with an antibody to a peptide in the C-terminal portion of BEHAB/brevican; the 50 kDa band is visualized with an antibody to a peptide in the N-terminal portion of BEHAB/brevican (see Materials and Methods). B, Western blots of surgical samples from neuropathologically diagnosed glioblastoma multiforme show 150 kDa full-length BEHAB/brevican and a 97 kDa proteolytic fragment. These bands are immunoreactive with the antibody to the C-terminal peptide; however, the antibody to the N-terminal peptide does not recognize the human protein.
- Fig. 2.
Transfected 9L cells express full-length BEHAB/brevican or the HABD. Western blots of cell homogenates (lanes 1–3) and conditioned media (lanes 4–6) are from transfected cells. 9L-GFP cell homogenates (lane 1) and conditioned media (lane 4) show no immunoreactivity for BEHAB/brevican or its cleavage products. 9L-BEHAB/brevican cell homogenates (lane 2) and conditioned media (lane 5) contain a 140 kDa-immunoreactive species. 9L-HABD cell homogenates (lane 3) and conditioned media (lane 6) contain a 50 kDa species. The immunoreactive species in conditioned media from both 9L-BEHAB/brevican and 9L-HABD transfectants are polydisperse (lanes 5, 6), possibly reflecting glycosylation.
- Fig. 3.
Full-length BEHAB/brevican and the HABD increase invasion and motility of 9L cells in vitro.A, Invasion in the Matrigel invasion assay is potentiated by the expression of either full-length BEHAB/brevican or the HABD. Two independent cell lines for each of the constructs (B/b-1 and B/b-2for 9L-BEHAB/brevican; HABD-1 and HABD-2for 9L-HABD) were assayed for invasion in the Matrigel invasion assay (see Materials and Methods). Invasion is expressed relative to that observed for the 9L-GFP transfectants. Both constructs markedly increased invasion. Invasion was equivalent when either fibronectin or hyaluronan was the attractant in the lower chamber. B, Motility in the absence of Matrigel is also increased by the expression of either full-length BEHAB/brevican or the HABD. As seen in the Matrigel assay, both constructs similarly increased motility in a Matrigel-independent assay. The degree of invasion or motility seen for all four transfected cell lines was statistically different from 9L-GFP cells at the p < 0.01 level. There was not a statistically significant difference between the behavior of 9LBEHAB/brevican versus 9L-HABD transfectants. Error bar indicates SEM.
- Fig. 4.
Intracranial tumors established from 9L-HABD, but not from 9L-BEHAB/brevican, cells show increased invasion into the surrounding brain. A,A′, Intracranial tumors (asterisks) derived from 9L-GFP cells grow as compact cell masses, with little infiltration into the surrounding brain. The border with the underlying thalamus (A′) is smooth, with very few clusters of cells seen beyond the border between the tumor and the normal brain.B, B′, Intracranial tumors (asterisks) derived from 9L-BEHAB/brevican cells showed identical behaviors to those of the control transfectants. These tumors also grew as compact cell masses, with little infiltration of the surrounding brain. Here again, very few cell clusters were observed in the normal brain adjacent to the tumor (B′).C, C′, Intracranial tumors (asterisks) derived from 9L-HABD cells showed a marked increase in invasive ability compared with the other two cell lines. Although the main tumor mass was a compactgroup of cells, many cell clusters were seen in the surrounding normal brain. The border of the 9L-HABD tumors with the underlying thalamus (C′) was often interrupted by peninsulas of cells extending out from the main tumor mass.
- Fig. 5.
Intracranial tumors established from 9L-BEHAB/brevican and 9L-HABD cells express full-length or HABD proteins. Western blots of tumors produced after intracranial injection of 9L-BEHAB/brevican (lane 1) or HABD (lane 2) transfectants are shown. In 9L-BEHAB/brevican tumors (lane 1), immunoreactivity for BEHAB/brevican is at 140 kDa. No evidence of a cleavage product is seen. In 9L-HABD tumors (lane 2), only a 50 kDa product is observed.
Tables
0.5–1.0 mm >1.0 mm 9L-GFP 1.67 ± 0.76 0.33 ± 0.33 9L-BEHAB/brevican 1.69 ± 0.77 0.15 ± 0.10 9L-HABD 8.33 ± 2.02 8.75 ± 3.70 The number of cell clusters located 0.5–1 mm and over 1 mm from the main tumor mass was counted for all of the tumors in this experiment (one random section per tumor; n = 6 independent tumors for 9L-GFP; n = 12 for 9L-BEHAB/brevican; and n = 14 for 9L-HABD). Two different transfected lines were used for each of the noncontrol transfectants. The HABD fragment markedly increased the number of cell clusters seen distant to the main tumor mass. The full-length BEHAB/brevican protein had no effect on cell infiltration into surrounding brain over that observed for the control 9L-GFP construct. The difference between the 9L-HABD versus the control 9L-GFP and 9L-BEHAB/brevican transfectants was statistically different at thep < 0.01 level. Data shown here are the grouped averages of the number of cell clusters ± SEM.
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