- Email: [email protected]
c 3T3 cells
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 2, March, pp. 447-457, 1983
Proteoglycans from the Substratum Adhesion Sites of MSV-Transformed BALB/c 3T3 Cells LAURA Department
of Microbiology, Received
J. MIKETO
Case Western September
Reserve
AND
LLOYD
University,
8, 1982; and in revised
A. CULP’
School form
of Medicine,
November
Clevelmd,
Ohio
&lo6
10, 1982
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(P-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 CLZB 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 GAG 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 highmolecular-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.
whereby tumor cells detach easily from a tumor mass and reimplant in target tissues. Therefore, an understanding of how tumorigenic and ultimately metastatic cells adhere to extracellular matrices in ways similar to or different from their “normal” cell counterparts is a central problem in tumor biology. As a model system, we have chosen to
The adherence properties of tumorigenie cells to either natural or artificial substrata (extracellular matrices) are significantly different from their “normal” cell counterparts (1, 2). This is particularly relevant to metastatic behavior (2), 1 Author dressed.
to whom
correspondence
should
be ad-
447
0003-9861/83/040447-11$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
448
MIKETO
study the biochemistry of adhesion of Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (3) to the simplest model substratum, the serum-coated (fibronectin-dependent) plastic tissue culture dish. These cells are highly tumorigenic in Balb/ c mice (3), are metastatic to the lung of mice (14), and display several properties reflecting fragile adherence to tissue culture substrata. Understanding of the molecular mechanism of Balb/c 3T3 adhesion to tissue culture substrata has been facilitated by studies on the composition and molecular organization of substratum-attached material (1,4), focal footpad adhesion sites left substratum-bound after EGTA’-mediated detachment of cells (5). This material is highly enriched in cell surface fibronectin, hyaluronic acid, other glycosaminoglycan (GAG)-containing proteoglycans (PG), and some cytoskeletal proteins (1, 6). A variety of studies on the composition, origin, and organization of substratum-attached material indicates that hyaluronic acid may function in the detachment of cells, whereas heparan sulfate PG functions in mediating at least one aspect of the adhesion of cells to a fibronectin-coated substratum (6, 7). Therefore, we have examined several biochemical properties of the GAGS and PG’s isolated from the substratum adhesive material of the highly tumorigenic Kirsten virus-transformed Balb/c 3T3 cells to correlate any qualitative and/or quantitative changes in their GAG species with their ease of detachability from the tissue culture substratum, their reluctance to spread over the substratum, and their noted lack of intracellular microfilament bundles (8). We have taken advantage of the methodologies developed for analyzing cartilage tissue proteoglycans (9) in these ’ Abbreviations used: EGTA, ethylene glycol bis(j3aminoethyl ether) N,W-tetraacetic acid; GAG, glycosaminoglycan; PG, proteoglycan; KiMSV, Kirsten murine sarcoma virus; L-SAM, long-term radiolabeled substratum-attached material; PBS, phosphate-buffered saline; R-SAM, reattaching substratum-attached material; SDS, sodium dodecyl sulfate; DNP-, 2,4-dinitrophenyl-; CS, chondroitin sulfate; HS, heparan sulfate.
AND CULP
studies, including the association-dissociation properties of these PG’s as evaluated by isopycnic CsCl density gradient centrifugation and gel filtration chromatography. These approaches have also proven useful in analyzing the properties of PG’s from tissue-cultured fibroblasts (lo-12), as well as the PG’s isolated from the substratum adhesion sites of parental Balb/c 3T3 cells (10, 13) from which these MSV-transformed cells were derived. MATERIALS
AND METHODS
CeU growth “Nonproducer” Balb/c 3T3 cells transformed by Kirsten murine sarcoma virus (K234 clone of KiMSV cells) (3) and Balb/c 3T3 cells were used between their 10th and 20th passages and cultured in Eagle’s minimum essential medium supplemented with four times essential amino acids and vitamins, 10% calf serum, 250 units/ml penicillin, and 0.25 mg/ ml streptomycin. They were incubated in an environment of 5% CO295% humidified air at a constant temperature of 37°C. Both cell types had similar doubling times of 17-19 h and were plated or harvested at similar densities for experiments (well before the cells became confluent). Radiolabeling and isolcstion of substratum-attached material. Long-term radiolabeled substratum-attached material (L-SAM) was prepared by inoculating 0.3-0.4 X lo6 cells into each of sixty-four lOO-mmdiameter tissue culture dishes containing 8 ml of complete medium for 24 h of attachment and growth (13). The medium was then removed and replaced with 8 ml of either complete medium containing 50 &i/ml Naes%IO, or 5pCi/ml[8Hlglucosamine hydrochloride. The cells were then incubated for 72 h, until they had reached 7080% confluence. The medium was aspirated off and the cell layer rinsed twice with phosphate-buffered saline (PBS). The cells were detached from the substratum by incubation with 0.5 mM EGTA in PBS for 30 min at 37°C on a gyratory shaker. To remove any cells still adherent, the cell suspension was gently pipetted over the surface of the dish. The radiolabeled cells were centrifuged, resuspended in fresh medium, and later used in preparing reattaching substratum-attached material (RSAM) by washing the cells twice with PBS and reinoculating them for 1 h into fresh dishes before a final detachment with EGTA. This l-h attachment provides cells with sufficient time to form new adhesion sites without any cell movement (5). Furthermore, it has previously been shown (18) that the vast majority of the GAG deposited in R-SAM was synthesized during the prior 72 h of growth in radiolabeling medium rather than during the l-h attachment period. In these experiments, the typical amounts of aminosugar-ra-
SUBSTRATUM
ADHESION
diolabeled material per dish were as follows: cell fraction, 1.1-1.4 X lO’cpm/dish; L-SAM, 1.4-1.6 X 106 cpm/dish; and R-SAM, 0.75-0.90 X ld cpm/dish. L-SAM remaining on the tissue culture substratum was rinsed three times with PBS and once with glassdistilled water before being extracted with 5 ml of 4 M guanidine hydrochloride (GuHCl) in buffer A (0.05 M sodium acetate containing the following protease inhibitors: 0.05 M benzamidine hydrochloride, 0.1 M 6aminohexanoic acid, and 0.01 M EDTA at pH 5.8). During the extraction, the dishes were incubated at 4°C on a rocking platform for 4 h which gives maximal extraction of protein or polysaccharide components. The L-SAM extract was then collected and concentrated by vacuum dialysis at 4°C. Reattaching substratum-attached material (RSAM) was prepared by inoculating all of the radiolabeled cells (see above) from the preparation of LSAM into thirty-two 100-mm tissue culture dishes containing 8 ml of complete medium. The cells were allowed to attach and spread for 1 h after which time the cells were detached with EGTA and the newly formed adhesion sites as R-SAM (5) extracted as previously described in the preparation of L-SAM. Gel filtration chromatography. L-SAM or R-SAM extracts were chromatographed on a Sepharose CL2B column (0.4 X 80 cm) under three buffer conditions: (a) semidissociated in 4 M GuHCl in buffer A, (b) reassociated by dialyzing the extract to a final concentration of 0.4 M GuHCl in buffer A for 24 h at 4’C [which allows proteoglycan complexes to reform (S)], and (c) completely dissociated in 0.2% SDS (in buffer B: 0.15 M sodium acetate, 1 mM MgClz, and 1 mM CaClz, pH 5.8). For all of the chromatography procedures, 0.5-ml fractions were collected and an aliquot of each fraction was assayed for radioactivity using scintillation counting. Void and inclusion volumes for each column were determined with blue dextran and DNP-glycine, respectively. Isopgmic fit9 gradient catrlfugatiorh Some of the L-SAM or R-SAM fractions extracted in 4 M GuHCl were brought to associative conditions as described above. Dissociative or associative extracts were aliquoted into polyallomer tubes already containing cesium chloride dissolved in buffer A and 4 M GuHCl or 0.4 M GuHCl, respectively, at a density of 1.54 or 1.63 g/ml, respectively (13). The tubes were centrifuged in a Beckman Type 65 rotor at 32,660 rpm for 72 h at 18°C. The tubes were then carefully withdrawn from the rotor and 0.5-ml fractions were collected from the bottom of the tube after needle puncture. An aliquot of each fraction was assayed for radioactivity using scintillation counting with correction for quenching. Densities of dissociative gradients were determined by weighing 100 ~1 of the gradient fractions, whereas densities of associative gradients were determined by refractive index. Enzyme digestions. Before enzymatic digestion,
OF
TRANSFORMED
CELLS
449
gradient peaks were pooled and cesium chloride was removed by extensive dialysis against glass-distilled water. For testicular hyaluronidase treatment, fractions were further dialyzed against buffer B for 24 h at 4°C and incubated with 10 pgg/ml testicular hyaluronidase for 4 h at 37°C. For Streptomyces hyaluronidase treatment, fractions were dialyzed against buffer C [O.l M sodium acetate, 150 m?d sodium chloride, 1 mM CaClz, and 1 mM MgClz (pH S.O)] for 24 h at 4°C and incubated with 10 TRU/ml Streptomyces hyaluronidase for 4 h at 37°C. For both enzymes, a control group received the same treatment as the enzyme-digested material with the exception that no enzyme was added. SDS was then added to all samples to a final concentration of 0.2% and the samples were applied to a Sepharose CL-6B column (1 X 80 cm) eluted with 0.2% SDS in buffer B. Fractions of 1.5 ml were collected and assayed for radioactivity by scintillation counting. Nitrous acid deamination To determine the heparan sulfate content of the gradient fractions, nitrous acid deamination was used as described previously (18). The gradient peaks were divided into experimental (treated with 1.8% sodium nitrite in 1.8 M acetic acid) and control (treated with acetic acid alone) halves and incubated for 80 min at room temperature. The reaction was terminated by addition of 2 M sodium sulfamate. All samples were brought to a final concentration of 0.2% SDS and chromatographed on a Sepharose CL-6B column eluted with 0.2% SDS in buffer B. Mate-rials. The following materials were purchased: [6-gi[lglucosamine hydrochloride and NazsSO, from Amersham Corporation; Pronase and Streptomyces hyaluronidase from Calbiochem Corporation; testicular hyaluronidase from Worthington Biochemical Corporation; cesium chloride and guanidine hydrochloride from Bethesda Research Laboratories; Sepharose CLdB and CL-6B from Pharmacia Fine Chemicals, Inc.; EGTA from Eastman Organic Chemicals; chondroitinases ABC and AC from Miles Laboratories, Inc.; calf serum from K. C. Biological, Inc.; plastic tissue culture dishes from Lux Scientific Company, reference standard GAGS kindly provided by Drs. Cifonelli and Mathews of the University of Chicago. RESULTS
GAG composition and extractability. The GAG distribution of the substratum attached material from KiMSV cells was first characterized by the methods of Rollins and Culp (18) and compared to the distribution in the adhesion sites of Balb/c 3T3 cells from which these transformed cells were derived (Table I). L-SAM and R-SAM
MIKETO
450 TABLE
I
GLYCOSAMINOGLYCANDISTRIBUTIONS Polysaccharide class*
Cell associated (%)
L-SAM (%)
R-SAM (%I
KiMSV
HS HA C6S C4S cos DS Total
72.0 10.9 ND” 5.7 1.8 9.6 100.0
39.0 24.3 5.2 15.2 6.3 10.0 100.0
25.6 20.8 10.7 18.2 8.2 16.5 196.0
Balb/c 3T3d
HS HA C6S c4s cos DS Total
48.8 17.6 0.8 5.7 3.1 24.0 106.66
26.3 22.7 2.3 22.4 23.6 2.7 100.0
80.2 4.5 1.6 8.0 4.8 0.9 loo.0
Cell line
’ Cells were radiolabeled for 72 h in medium containing [SHlglucosamine after which L-SAM and RSAM were prepared (Materials and Methods) and quantitatively solubilized with SDS. Specific GAG classes were identified as described by Rollins and Culp (18). These data are representative of three separate determinations. * HS, Heparan sulfate; HA, hyaluronic acid, C-6-S, chondroitin g-sulfate; C-4-S, chondroitin 4-sulfate; COS, unsulfated chondroitin; DS, dermatan sulfate. The standard error in measurement of these numbers for separate sets of samples varied from +6% for the high-concentration GAGS to +13% for the low-concentrated GAGS. ‘None detected. d These data are taken from Ref. (18) for comparison with the data of the KiMSV cells being analyzed in this study.
from KiMSV cells have similar distributions of the various GAG components. Both adhesion sites are depleted in the amount of heparan sulfate and are enriched for hyaluronic acid and the chondroitin sulfate classes when compared to the cell-associated material (that is, EGTA-detached cells). Substratum-attached material from long-term growing or reattaching Balb/c 3T3 cells differ, however, in their GAG composition, with the R-SAM containing much more heparan sulfate and less hyaluronic acid and chondroitin sulfate than
AND CULP
the L-SAM. The GAG compositions of the L-SAMs from both cells are similar, whereas the R-SAMs differ significantly. The much higher amounts of hyaluronic acid and chondroitin sulfates of KiMSV RSAM sites correlates with the indication (6) that hyaluronic acid and/or the chondroitin sulfates are involved in detachment of cells from the substratum; newly attaching KiMSV cells are much easier to detach during EGTA treatment than newly attaching 3T3 cells (data not shown). In order to study the reassociation patterns of the GAGS and PG’s in the adhesion sites, the substratum-attached material was extracted with 4 M guanidine hydrochloride (GuHCl) under conditions that do not irreversibly denature proteoglycan species in order to further study their potential to reassociate into highmolecular-weight complexes (9). The percentages of the adhesion site protein and polysaccharide extracted with 4 M GuHCl are presented in Table II for comparison with SDS-solubilized material, which quantitatively removes L-SAM or R-SAM (13). GuHCl, 4 M, extracted equal amounts of KiMSV L-SAM radiolabeled with [3H]glucosamine or ?SO,2-; however, significantly more ?SO$--labeled material was extracted from the reattaching adhesion sites than [3H]glucosamine-labeled material. In comparison to Balb/c 3T3 cells, KiMSV adhesion sites were more labile to the 4 M GuHCl treatment. Various properties of the KiMSV GAG-containing moieties were then examined by gel filtration chromatography or isopycnic density gradient analyses. Chrmatography of extracts under associative or dissociative conclitiona Substratum-attached material (long-term generated and extracted with 4 M GuHCl) was dialyzed against 0.4 M GuHCl to permit reassociation of molecules into complexes or against 0.2% SDS to completely dissociate aggregates (13). Chromatography of the extracts on Sepharose CL-2B columns eluted with the same buffers is shown in Fig. 1. None of the sulfated GAG appeared at the void volume of the column eluted with 4 M GuHCl (Fig. 1A) or with SDS (Fig. 1C) indicating that high-molecular-weight
SUBSTRATUM
ADHESION
OF TABLE
GuHCl
TRANSFORMED II
EXTRACTABILI~ Radioactivity
Cell type
Cell fraction
KiMSV
Balb/c
3T3
451
CELLS
[SH]Glucosamine
(% of total)* N&?!30,
L-SAM R-SAM
54 35
53 63
L-SAM R-SAM
46 21
NDc ND
a L-SAM or R-SAM radiolabeled with [*Hlglucosamine of Naza?SO1 were prepared as described under Materials and Methods. The dishes bearing adhesion sites were then extracted with 4 M GuHCl as described under Materials and Methods. An aliquot of the GuHCl extract was assayed for radioactivity and compared with an equal aliquot of an SDS extract of a parallel set of dishes (SDS quantitatively solubilizes material from adhesion sites); corrections were made in all samples for quenching. * Calculated by: radioactivity solubilized with GuHCl f radioactivity solubilized with SDS X 166. ‘Not determined. A
57
4M GuHCl
II
,,-I
I IO
1 1
09 OS
IO
20 30 40 FRACTION NUMBER
50
60
FIG. 1. Gel filtration chromatography of L-SAM under associative and dissociative conditions. PHIGlucosamineor =SOg--radiolabeled KiMSV L-SAMs were isolated as described under Materials and Meth-
complexes were not present under these dissociative conditions. However, significant sulfated GAG appeared at V, after dialysis aginst 0.4 M GuHCl (Fig. lB), demonstrating the formation of such complexes. This was further shown by pooling the VO-eluting material in Fig. lB, dialyzing it against SDS, and rechromatographing on an SDS-eluted Sepharose CL-2B column; all of the material shifted well into the included region of the column (K,, = 0.5-0.7). Furthermore, the 4 M GuHCl extract consistently showed intermediatesized sulfated complexes (fractions 20-30 of Fig. 1A) which were not present in SDS extracts (Fig. 1C). A sizable amount of gluods. A portion of the L-SAM extracted in 4 M GuHCl in buffer A was directly chromatographed on a Sepharose CL-2B column eluted with 4 M GuHCl in buffer A (A). The remaining L-SAM was brought to associative conditions by extensive dialysis against 0.4 M GuHCl in buffer A and a sample chromatographed on a Sepharose CL-2B column eluted with 0.4 M GuHCl in buffer A (B). A final sample of L-SAM was further dialyzed against buffer B to remove all guanidine hydrochloride and brought to dissociative conditions by extensive dialysis against 0.2% SDS in buffer B (C). Fractions were analyzed for eH (0) or as (0) radioactivity as described under Materials and Methods. Void and inclusion volumes were determined using blue dextran and dinitrophenylated glycine. Only the ordinate on the right in A is cpm ?S X 103, whereas the ordinates in B and C are cpm (without any multiplication factor).
452
MIKETO
AND
cosamine-radiolabeled (nonsulfated) material persisted at V, of Fig. 1C in SDS and was shown to be hyaluronic acid by its sensitivity to Streptmgces hyaluronidase (data not shown). These experiments indicate that a portion of the sulfated GAG extracts from L-SAM can reaggregate into large complexes which are partially sensitive to 4 M GuHCl and completely sensitive to SDS. Similar analyses were made of R-SAM extracts (Fig. 2). There was a significant amount of sulfated GAG at the void volume of the column eluted with 0.4 M GuHCl (Fig. 2B) that was not apparent in 4 M GuHCl extracts (Fig. 2A), similar to the situation with L-SAM in Fig. 1. On the other hand, the R-SAM extracts (4 M GuHCl) contain very little intermediatesized aggregates (compare fractions 20-30 50
A.
4M
1000 1
GuHCl
4.5
900
2.5 _
em 700 - 600 500
2.0 -
- 400
4.0 "0 i
3.5 . 3.0 -
Vi 4
1.5 .
xx)
1.0 5F 0.5
200 100
M f 4.5 6. 7
0.4M
:: E
GuHCl
;; 4.0 4 3.5
800
3.0
600;
In, * z _o ” ,I
F6
‘;
7007 8
2.5
500
,,*
2.0
400
1.5
3cxJ
; c)
1.0
200
0.5
loo
FRACTION
NUMBER
FIG. 2. Gel filtration chromatography of R-SAM under associative and dissociative conditions. [“HIGlucosamineor ?S@--radiolabeled R-SAM samples were treated and analyzed as described in the legend to Fig. 1 for chromatography on columns of Sepharose CL-2B.
CULP
A.
ASSOCIATIVE -50 . 4.5
25.
1,.
I.0 20
. .-. 3.0 40 5D 61) 70 8.0 9D
IO0
I
FIG. 3. Isopycnic density gradient centrifugation of L-SAM. L-SAM radiolabeled with ~lglucosamine (O l ), or Naaa6S0, (0 - - - 0) was centrifuged on CsCl gradients under associative (A) or dissociative conditions (B) as described under Materials and Methods. Associative L-SAM was prepared by dialyzing L-SAM harvested in 4 M GuHCl against 0.4 M GuHCl in buffer A for ‘72 h. L-SAM extracted in 4 M GuHCl was directly applied to the CsCl gradient for centrifugation under dissociative conditions. Fractions of 0.5 ml were collected from the bottom of gradient tubes and assayed for ‘H (O-O), “S (0 - - - 0), or density (-) as described under Materials and Methods.
of Fig. 2A with Fig. 1A). GuHCl, 4 M, extracts of R-SAM also contained much less hyaluronic acid at the V, region of Figs. 2A or C when compared to L-SAM extracts. Density gradient centr&gaticm of extracts mder associative w dissociative umditims. When long-term generated substratum-attached material was centrifuged to equilibrium in a CsCl gradient under associative conditions, three peaks of radiolabeled material were observed (Fig. 3A)-GJ, GA2, and GA3 at densities of 1.67-1.71, 1.62-1.65, and 1.6-1.62 g/cm3, respectively. Although all three compo-
SUBSTRATUM
ADHESION
nents contain [3HJglucosamine-radiolabeled material, only GA1 and GA3 contained sulfated GAG. Similar peaks were observed with the associative extracts of Balb/c 3T3 L-SAM by Garner and Culp (13). Under dissociative conditions, KiMSV extracts generated a sizable pool of material at the top of the gradient (Go2 in Fig. 3B) and a small amount of sulfated GAG at the bottom of the gradient (Gnl). Similar analyses of R-SAM extracts on density gradients are shown in Fig. 4, and there are significant differences with the L-SAM profiles of Fig. 3. There is virtually no GA2 material in R-SAM centrifuged under associative conditions (Fig. 4A) and much less G,3; most of the material is highly sulfated and dense, appearing in the bottom fractions of the gradient in GAl. Another difference is noted when R-SAM is analyzed under dissociative conditions (Fig. 4B)-much of the sulfated material remains highly dense and does not shift to the top of the gradient as observed in L-SAM (Fig. 3B). Although analyses for specific GAGS demonstrate very similar distributions from both L-SAM and RA
B
ASSOCIATIVE
DISSOCIATIVE
1.0 20 30 40 50 60 70 80 VOLUME (ml)
90 10.0
FIG. 4. Isopycnic density gradient centrifugation of R-SAM [8H]Glucosamine- or Naz%@--radiolabeled R-SAM samples were prepared and analyzed on CsCl associative or dissociative gradients as described in the legend to Fig. 3.
OF TRANSFORMED 1600
I400
A. L-SAM (GlcN)
453
CELLS
Gnl
1200 5 1000 5L? so0 L 600 \ E 400 8 -?Q 200 x
IO
20
30
FRACTION
40
50
60
70
NUMBER
FIG. 5. Sensitivity of aminosugar-radiolabeled GJ to hyaluronidase digestion. IjHlglucosamine-radiolabeled L-SAM or R-SAM were extracted with 4 M GuHCl, dialyzed against 0.4 M GuHCl, and centrifuged on an associative cesium chloride gradient (Fig. 3A or 4A) as described under Materials and Methods. Fractions from the GJ peaks were pooled and treated with testicular hyaluronidase (A A), Streptomyces hyaluronidase (A---A) or no enzyme as a control (O 0). After enzymatic digestions, samples were chromatographed on a Sepharose CL-6B column eluted with SDS buffer as described under Materials and Methods. (A) PH]Glucosamine L-SAM G*l; (B) [8Hlglucosamine R-SAM GJ.
SAM (Table I), the 4 M GuHCl extracts of these adhesive materials behave quite differently upon density gradient analyses. Biochemical characterization of spe&$c GAG’s in density gradient peaks. We then chose to identify specific GAG moieties in the density gradient fractions of L-SAM and R-SAM. Because of the paucity of material, we were forced to use sensitivity to specific GAG hydrolases or chemical agents, along with shifts in material upon gel filtration chromatography, as assays for specific GAG classes. A comparison of the enzymatic sensitivities of associative extract GA1 pools of L-SAM or R-SAM is displayed in Fig. 5 for aminosugar-radiolabeled material. The control digest of L-SAM (Fig. 5A) generates two well-separated entities upon chromatography on Sepharose CLGB in an SDS-containing buffer-one minor moiety
454
MIKETO
AND
eluting just after V. and a major component with K,, = 0.45-0.55. The minor moiety is completely sensitive to Strepb myces hyaluronidase, demonstrating that this is HA, and some of the major component is also sensitive to the Streptomyces enzyme, although more of this material is sensitive to testicular hyaluronidase (chondroitin sulfate-containing moieties). R-SAM GA1 is considerably different (Fig. 5B), with virtually no HA (Streptomyces hyaluronidase insensitivity) but comparable amounts of chondroitin sulfate to L-SAM GAl. Treatment of sulfate-radiolabeled material from GA1 with either testicular hyaluronidase or nitrous acid can distinguish heparan sulfate from chondroitin sulfate entities (Fig. 6). From L-SAM, approximately 30% of the major sulfated peak is sensitive to the testicular enzyme (CS)
20
30 FRACTION
40
60
60
7'0
NUMBER
FIG. 6. Sensitivity of ‘%Oz--radiolabeled G*l to various treatments. L-SAM and R-SAM from “SO:--radiolabeled KiMSV cells were extracted with 4 M GuHCl, dialyzed against 0.4 M GuHCl, and centrifuged on an associative cesium chloride gradient (Figs. 3A and 4A) as described under Materials and Methods. Fractions containing GJ were pooled and split into three samples. The first sample was digested with testicular hyaluronidase (A A), while the second sample was treated with nitrous acid (0 - - - 0). The third sample was left untreated as a control (0-O). All three samples were chromatographed on a Sepharose CL-6B column eluted with an SDS buffer as described under Materials and Methods. (A) &SO:--radiolabeled L-SAM GAl; (B) “SO:--radiolabeled R-SAM Gnl.
CULP
4.or
‘A2
1
3.5 p 3.0 Y g 25E " 2.0 72 ; 1.5Y " & l.On 0.51
IO
20
30
FRACTION
40
50
60
NUMBER
FIG. 7. Sensitivity of GA2 to Streptcrm~ces hyaluronidase digestion. pH]Glucosamine-radiolabeled L-SAM was centrifuged under associative conditions as described under Materials and Methods and Fig. 3A. Fractions from peak GA2 were pooled and split into two parts after dialysis against digestion buffers. One sample was treated with Streptom~ces hyaluronidase (0 - - - 0) while the other sample was left untreated as a control (0 0). Both samples were chromatographed on a Sepharose CL-6B column eluted with SDS-containing buffer.
while the remaining 70% is sensitive to breakdown with nitrous acid (HS). Similar sensitivities were observed for R-SAM GA1 (Fig. 6B), with the single exception that a higher proportion of the major peak was HONO-sensitive. The results of Fig. 5 and 6 indicate that the dense material banding at the bottom of associative gradients is principally HS proteoglycan, with a smaller amount of CS proteoglycan and, in the case of L-SAM, some hyaluronic acid. Since HA alone bands at much lighter densities in these gradients (see below), this “dense” HA must be complexed with one or more of the proteoglycan species. GA2 from L-SAM (Fig. 3A) is entirely HA, as determined by its complete sensitivity to Streptomgces hyaluronidase (Fig. 7). Similarly, when nonradiolabeled HA was analyzed under identical conditions, it bands only in the GA2 region of gradients and not in GA1 or GA3 regions (data not shown). Therefore, there is a large excess of HA in the 4 M GuHCl extracts of L-SAM (and no GA2-containing HA in the 4 M GuHCl extract of R-SAM) and this moiety should not be a limiting factor in
SUBSTRATUM
ADHESION
OF
formation of supramolecular aggregates of these extracts (9). The content of GAG in GA3 was then determined. Sensitivity to enzyme digestion of aminosugar-radiolabeled material is shown in Fig. 8. The control digest of LSAM generates a large peak at Vi and a smaller included peak when eluted from Sepharose CL-6B columns in an SDS-containing buffer (Fig. SA). The Vi material is completely sensitive to Streptomvces hyaluronidase showing that this is HA. This could be some cross-contaminating HA from the GA2 peak (see Fig. 3A) or a separate class of HA. Interestingly, R-SAM also contained this class of HA in GA3 (Fig. SB), although R-SAM ccmtains no GA.2peak; 2.2 2.0
I.6 f
A
L-SAM (GlcN)
GA3
I.0 g
0.6
2
0.6
E
0.4
ho.2 0 P-l E2 x z g
I.6
g
1.2
14
IO
20
FRACTION
30
40
50
60
NUMBER
FIG. 8. Sensitivity of GA3 to hyaluronidase digestion. GA3 fractions (see Figs. 3A and 4A) of mlucosamine-radiolabeled L-SAM or R-SAM were pooled separately, dialyzed against digestion buffers, and digested with testicular hyaluronidase (A A) or Streptomgca hyaluronidase (A - - - A). After enzymatic digestions, samples were chromatographed on Sepharose CL-6B columns eluted with SDS-containing buffers for comparison with elution patterns of an undigested control (0-O). (A) [4I]Glucosamine-radiolabeled L-SAM Gn3; (B) [SHlglucosamine-radiolabeled R-SAM Gn3.
TRANSFORMED
CELLS
5
16-
A. L-SAM (GlcN)
I= 2 &
14. 12.
V, \
g
IO-
455
G,l
0.6 -
FIG. 9. Sensitivity of L-SAM Go1 to various treatments. Gnl from L-SAM extracts after dissociative analysis on a cesium chloride gradient (Fig. 3B) was pooled, dialyzed against the appropriate buffer, and treated with either testicular hyaluronidase (A A), nitrous acid (0 -. - 0), or left untreated (O 0). Samples were then chromatographed on a Sepharose CL-6B column in an SDS-containing buffer as described under Materials and Methods. (A) [SHlglucosamine-radiolabeled L-SAM Gnl; (B) %O:--radiolabeled L-SAM Gnl.
it appears likely then that the HA in GA3 is a separate class of HA with possibly glycoprotein bound to it to give it a much lighter density. A very small amount of the included material of L-SAM GA3 (Fig. SA) was sensitive to testicular but not Streptomyces hyaluronidase, indicating some CS. This was not the case for R-SAM GA3. All of the sulfate-radiolabeled GA3 from L-SAM was sensitive to testicular hyaluronidase but not to HONO, demonstrating again some CS in GA3 but no HS (data not shown). It appears that most of the included aminosugar-radiolabeled material in GA3 is glycoprotein. The small amount of Gnl from L-SAM dissociative gradients which persists at high density was tested for its sensitivity (Fig. 9). The high-molecular-weight aminosugar-radiolabeled material at VO is sensitive to testicular hyaluronidase (Fig. 9A) but not to Streptmyces hyaluronidase and therefore must be proteoglycan-containing CS sequences (Pronase digestion shifts this material into included regions
456
MIKETO
of the profile) (data not shown). Approximately one-half of the sulfate-radiolabeled Gnl is CS (sensitive to testicular hyaluronidase) while the remainder is HS (sensitive to HONO) (Fig. 9B). R-SAM Gnl content is considerably different (Fig. 10). The vast majority of the sulfate- or aminosugar-(not shown) radiolabeled material is HS with only a minor fraction of CS. DISCUSSION
These results have characterized in a preliminary fashion some properties of proteoglycans from the newly formed or “matured” substratum adhesion sites of a metastatic malignant derivative of Balb/ c. 3T3 cells, KiMSV (3, 14). Considerable evidence is accumulating that heparan sulfate proteoglycans play a direct role in forming substratum adhesion sites whereas hyaluronic acid may function in the detachment of cells (6, 7, 15, 16, 19). The ability of both cellular and plasma fibronectins to bind to HS sequences while only the aggregated form of cellular fibronectin binds to HA is consistent with these proposals (1’7). It, therefore, becomes imGDl
R-SAM
9 ]b
(SO&')
Es o 0.8 5x 0.6 'I* 8 0.4 x 0.2 IO
20
FRACTION
30
40
50
60
NUMBER
FIG. 10. Sensitivity of R-SAM Gnl to testicular hyaluronidase or nitrous acid. Gnl fractions from %3@--radiolabeled R-SAM extracts were pooled, dialyzed against the appropriate buffer, and split into three samples. One sample was digested with testicular hyaluronidase (A A), the second treated with nitrous acid (0 -a - 0), and the third left untreated as a control (0 0). All three samples were chromatographed on a Sepharose CL-6B column eluted with an SDS-containing buffer as described under Materials and Methods.
AND
CULP
portant to biochemically characterize the hyaluronate and various GAG-containing proteoglycans from the substratum-attached material of these cells in order to better understand their potential functions. The data reported herein demonstrate some significant differences in the GAG moieties from the adhesion sites of KiMSV cells when compared to the parental Balb/c 3T3 cells (13). In particular, the RSAM of KiMSV cells contains much more HA and CS than the R-SAM of 3T3 cells; this may reflect the considerable ease of detachment of KiMSV cells from the substratum under all culture conditions. However, much of the HA in KiMSV RSAM is nonextractable with 4 M GuHCl but becomes extractable with “maturing” of these sites into L-SAM. Furthermore, the GA3 density gradient pool of KiMSV cells contained considerable “light density” HA but no HS; Balb/c 3T3 GA3 contained very little HA but significant amounts of HS (13). A much smaller proportion of proteoglycan material extractable with 4 M GuHCl from KiMSV L-SAM or R-SAM could reaggregate when dialyzed against associative buffers (as analyzed by gel filtration chromatography) when compared to 3T3 cells (13). On the other hand, KiMSV extracts in either 4 M or 0.4 M GuHCl contained intermediatesized aggregates sensitive to SDS; 3T3 extracts generally contained only high-molecular-weight aggregates excluded from Sepharose CLZB columns (13). All of these data indicate appreciable quantitative and/or qualitative differences in the proteoglycan moieties from the adhesive material of these two cell types that display very different growth potentials in animals and significantly different adhesive behaviors on tissue culture substrata. In agreement with the previous data of Garner and Culp (13) with Balb/c 3T3 cells, there are differences in the 4 M GuHCl extracts of newly formed adhesion sites (RSAM) or “matured” sites (L-SAM) of KiMSV cells. R-SAM contains much less GuHCl-extractable HA than L-SAM as demonstrated with the I’,,-eluting material from gel filtration columns (Figs. 1 and 2) or the GA2 band of isopycnic density
SUBSTRATUM
ADHESION
gradients (Figs. 3 and 4). The presence of HA in GJ of L-SAM associative gradients is reminiscent of the HA:chondroitin sulfate proteoglycan aggregates found in cartilage (9); similar aggregates have been found in the reassociated extracts of tissue cultured human fibroblasts (11, 12). Whether the HA in this high-density material from L-SAM (but not R-SAM) is truly bound to CS-PG remains to be proven. Although R-SAM contains no intermediate-density GA2, whereas L-SAM contains much of this material, R-SAM contains considerable “light density” HA in a smaller total pool of GA3. L-SAM contained a small amount of HA in GA3, which could possibly be explained by cross-contamination by the large amount of HA in L-SAM GA2. The nature of the material which binds to HA under these associative conditions to make it “light” is of considerable interest. Another important difference between R-SAM and L-SAM extracts is the behavior of heparan sulfate moieties. The vast majority of the HS in L-SAM shifts to the top of dissociative gradients, whereas most of the HS in R-SAM remains in the high density fractions of dissociative gradients. One explanation for this derives from the data of Garner and Culp (13), namely, that GD1 HS occurs as large proteoglycans, whereas L-SAM Go3 HS occurs as free HS chains. This would indicate that newly formed adhesion sites contain principally HS-PG that is later catabolized by some unknown mechanism into free HS chains. Alternatively, the light-density HS may be in the form of a proteoglycan with a hydrophobic region of the core protein which allows these to aggregate into lowdensity complexes (20); R-SAM and L-SAM would then have very different distributions of such a membrane-associated proteoglycan and there would be metabolic conversion of one form of the proteoglycan into the other form. Clearly, more information will be required to prove or disprove such a metabolic conversion process in the adhesion sites of cells. This would raise the important issue as to whether metastatic and invasive cell types have a greater capacity to catabolize the proteoglycans in their substratum adhesion sites
OF
TRANSFORMED
CELLS
457
when compared to their normal cell counterparts, an issue that will be directly addressed in future studies. ACKNOWLEDGMENTS This work was supported by a National Institutes of Health Research Grant AM25646. The authors wish to thank Dr. Judy Garner and Michael Lark for many helpful discussions. REFERENCES 1. CULP, L. A. (1978) in Current Topics in Membranes and Transport (Juliano, R. L., and Rothstein, A., eds.), Vol. 11, pp. 327-396, Academic Press, New York. 2. POSTE, G., AND FIDLER, I. J. (1980) Nature (London) 283, 139-146. 3. STEPHENSON, J. R., AND AARONSON, S. A. (1972) J. Exp. Mea! 135.503-514. 4. C~LP, L. A., MURRAY, B., AND ROLLINS, B. J. (1979) J. Supram Struck. 11.401-427. 5. ROSEN, J. J., AND CULP, L. A. (1977) Exp. Cell Res 107, 139-149. 6. ROLLINS, B. J., CATHCART, M. K., AND CLJLP, L. A. (1982) in The Glycoconjugates (Horowitz, M. I., ed.), Vol. III, pp. 289-329, Academic Press, New York. 7. LATERRA, J., SILBERT, J. K., AND CULP, L. A, (1983) J. Cell Biol 96, in press. 8. ALTENBURG, B. C., SOMERS, K., AND STEINER, S. (1976) Cancer Res. 36,251-260. 9. HASCALL, V. C. (1977) J. Szqraml Struet. 7, lOl120. 10. ROLLINS, B. J., AND CIJLP, L. A. (1979) Biodwmistry 18.5621-5629. 11. COSTER, L., CARLSTEDT, I., AND MALMSTROM, A. (1979) Biochem J. 183.669-677. 12. VOGEL, K. G., AND PETERSON, D. W. (1981) J. Bid Ch 256,13235-13242. 13. GARNER, J. A., AND CULP, L. A. (1981) Biochxmistry 20, 7350-7359. 14. NICOLSON, G., BRIJNSON, K. W. B., AND FIDLER, I. J. (1978) Cancer Res. 38.4105-4111. 15. BARNHART, B. J., Cox, S. H., AND KRAEMER, P. M. (1979) Exp. Cell Rex 119, 327-332. 16. RICH, A. M., PEARLSTEIN, E., WEISSMANN, G., AND HOFFSTEIN, S. T. (1981) Nature (London) 293, 224-226. 17. LATERRA, J., AND C~LP, L. A. (1982) J. Bid Ch 257,719-726. 18. ROLLINS, B. J., AND C~LP, L. A. (1979) Biochanistry 18.141-148. 19. KNOX, P., AND WELLS, P. (1979) J. CeU Sci 40,7789. 20. KJELLEN, L., PETTERSSON, I., AND HOOK, M. (1981) Proa Nat. Acad Sci USA 78, 5371-5375.
Proteoglycans from the Substratum Adhesion Sites of MSV-Transformed BALB/c 3T3 Cells LAURA Department
of Microbiology, Received
J. MIKETO
Case Western September
Reserve
AND
LLOYD
University,
8, 1982; and in revised
A. CULP’
School form
of Medicine,
November
Clevelmd,
Ohio
&lo6
10, 1982
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(P-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 CLZB 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 GAG 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 highmolecular-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.
whereby tumor cells detach easily from a tumor mass and reimplant in target tissues. Therefore, an understanding of how tumorigenic and ultimately metastatic cells adhere to extracellular matrices in ways similar to or different from their “normal” cell counterparts is a central problem in tumor biology. As a model system, we have chosen to
The adherence properties of tumorigenie cells to either natural or artificial substrata (extracellular matrices) are significantly different from their “normal” cell counterparts (1, 2). This is particularly relevant to metastatic behavior (2), 1 Author dressed.
to whom
correspondence
should
be ad-
447
0003-9861/83/040447-11$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
448
MIKETO
study the biochemistry of adhesion of Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (3) to the simplest model substratum, the serum-coated (fibronectin-dependent) plastic tissue culture dish. These cells are highly tumorigenic in Balb/ c mice (3), are metastatic to the lung of mice (14), and display several properties reflecting fragile adherence to tissue culture substrata. Understanding of the molecular mechanism of Balb/c 3T3 adhesion to tissue culture substrata has been facilitated by studies on the composition and molecular organization of substratum-attached material (1,4), focal footpad adhesion sites left substratum-bound after EGTA’-mediated detachment of cells (5). This material is highly enriched in cell surface fibronectin, hyaluronic acid, other glycosaminoglycan (GAG)-containing proteoglycans (PG), and some cytoskeletal proteins (1, 6). A variety of studies on the composition, origin, and organization of substratum-attached material indicates that hyaluronic acid may function in the detachment of cells, whereas heparan sulfate PG functions in mediating at least one aspect of the adhesion of cells to a fibronectin-coated substratum (6, 7). Therefore, we have examined several biochemical properties of the GAGS and PG’s isolated from the substratum adhesive material of the highly tumorigenic Kirsten virus-transformed Balb/c 3T3 cells to correlate any qualitative and/or quantitative changes in their GAG species with their ease of detachability from the tissue culture substratum, their reluctance to spread over the substratum, and their noted lack of intracellular microfilament bundles (8). We have taken advantage of the methodologies developed for analyzing cartilage tissue proteoglycans (9) in these ’ Abbreviations used: EGTA, ethylene glycol bis(j3aminoethyl ether) N,W-tetraacetic acid; GAG, glycosaminoglycan; PG, proteoglycan; KiMSV, Kirsten murine sarcoma virus; L-SAM, long-term radiolabeled substratum-attached material; PBS, phosphate-buffered saline; R-SAM, reattaching substratum-attached material; SDS, sodium dodecyl sulfate; DNP-, 2,4-dinitrophenyl-; CS, chondroitin sulfate; HS, heparan sulfate.
AND CULP
studies, including the association-dissociation properties of these PG’s as evaluated by isopycnic CsCl density gradient centrifugation and gel filtration chromatography. These approaches have also proven useful in analyzing the properties of PG’s from tissue-cultured fibroblasts (lo-12), as well as the PG’s isolated from the substratum adhesion sites of parental Balb/c 3T3 cells (10, 13) from which these MSV-transformed cells were derived. MATERIALS
AND METHODS
CeU growth “Nonproducer” Balb/c 3T3 cells transformed by Kirsten murine sarcoma virus (K234 clone of KiMSV cells) (3) and Balb/c 3T3 cells were used between their 10th and 20th passages and cultured in Eagle’s minimum essential medium supplemented with four times essential amino acids and vitamins, 10% calf serum, 250 units/ml penicillin, and 0.25 mg/ ml streptomycin. They were incubated in an environment of 5% CO295% humidified air at a constant temperature of 37°C. Both cell types had similar doubling times of 17-19 h and were plated or harvested at similar densities for experiments (well before the cells became confluent). Radiolabeling and isolcstion of substratum-attached material. Long-term radiolabeled substratum-attached material (L-SAM) was prepared by inoculating 0.3-0.4 X lo6 cells into each of sixty-four lOO-mmdiameter tissue culture dishes containing 8 ml of complete medium for 24 h of attachment and growth (13). The medium was then removed and replaced with 8 ml of either complete medium containing 50 &i/ml Naes%IO, or 5pCi/ml[8Hlglucosamine hydrochloride. The cells were then incubated for 72 h, until they had reached 7080% confluence. The medium was aspirated off and the cell layer rinsed twice with phosphate-buffered saline (PBS). The cells were detached from the substratum by incubation with 0.5 mM EGTA in PBS for 30 min at 37°C on a gyratory shaker. To remove any cells still adherent, the cell suspension was gently pipetted over the surface of the dish. The radiolabeled cells were centrifuged, resuspended in fresh medium, and later used in preparing reattaching substratum-attached material (RSAM) by washing the cells twice with PBS and reinoculating them for 1 h into fresh dishes before a final detachment with EGTA. This l-h attachment provides cells with sufficient time to form new adhesion sites without any cell movement (5). Furthermore, it has previously been shown (18) that the vast majority of the GAG deposited in R-SAM was synthesized during the prior 72 h of growth in radiolabeling medium rather than during the l-h attachment period. In these experiments, the typical amounts of aminosugar-ra-
SUBSTRATUM
ADHESION
diolabeled material per dish were as follows: cell fraction, 1.1-1.4 X lO’cpm/dish; L-SAM, 1.4-1.6 X 106 cpm/dish; and R-SAM, 0.75-0.90 X ld cpm/dish. L-SAM remaining on the tissue culture substratum was rinsed three times with PBS and once with glassdistilled water before being extracted with 5 ml of 4 M guanidine hydrochloride (GuHCl) in buffer A (0.05 M sodium acetate containing the following protease inhibitors: 0.05 M benzamidine hydrochloride, 0.1 M 6aminohexanoic acid, and 0.01 M EDTA at pH 5.8). During the extraction, the dishes were incubated at 4°C on a rocking platform for 4 h which gives maximal extraction of protein or polysaccharide components. The L-SAM extract was then collected and concentrated by vacuum dialysis at 4°C. Reattaching substratum-attached material (RSAM) was prepared by inoculating all of the radiolabeled cells (see above) from the preparation of LSAM into thirty-two 100-mm tissue culture dishes containing 8 ml of complete medium. The cells were allowed to attach and spread for 1 h after which time the cells were detached with EGTA and the newly formed adhesion sites as R-SAM (5) extracted as previously described in the preparation of L-SAM. Gel filtration chromatography. L-SAM or R-SAM extracts were chromatographed on a Sepharose CL2B column (0.4 X 80 cm) under three buffer conditions: (a) semidissociated in 4 M GuHCl in buffer A, (b) reassociated by dialyzing the extract to a final concentration of 0.4 M GuHCl in buffer A for 24 h at 4’C [which allows proteoglycan complexes to reform (S)], and (c) completely dissociated in 0.2% SDS (in buffer B: 0.15 M sodium acetate, 1 mM MgClz, and 1 mM CaClz, pH 5.8). For all of the chromatography procedures, 0.5-ml fractions were collected and an aliquot of each fraction was assayed for radioactivity using scintillation counting. Void and inclusion volumes for each column were determined with blue dextran and DNP-glycine, respectively. Isopgmic fit9 gradient catrlfugatiorh Some of the L-SAM or R-SAM fractions extracted in 4 M GuHCl were brought to associative conditions as described above. Dissociative or associative extracts were aliquoted into polyallomer tubes already containing cesium chloride dissolved in buffer A and 4 M GuHCl or 0.4 M GuHCl, respectively, at a density of 1.54 or 1.63 g/ml, respectively (13). The tubes were centrifuged in a Beckman Type 65 rotor at 32,660 rpm for 72 h at 18°C. The tubes were then carefully withdrawn from the rotor and 0.5-ml fractions were collected from the bottom of the tube after needle puncture. An aliquot of each fraction was assayed for radioactivity using scintillation counting with correction for quenching. Densities of dissociative gradients were determined by weighing 100 ~1 of the gradient fractions, whereas densities of associative gradients were determined by refractive index. Enzyme digestions. Before enzymatic digestion,
OF
TRANSFORMED
CELLS
449
gradient peaks were pooled and cesium chloride was removed by extensive dialysis against glass-distilled water. For testicular hyaluronidase treatment, fractions were further dialyzed against buffer B for 24 h at 4°C and incubated with 10 pgg/ml testicular hyaluronidase for 4 h at 37°C. For Streptomyces hyaluronidase treatment, fractions were dialyzed against buffer C [O.l M sodium acetate, 150 m?d sodium chloride, 1 mM CaClz, and 1 mM MgClz (pH S.O)] for 24 h at 4°C and incubated with 10 TRU/ml Streptomyces hyaluronidase for 4 h at 37°C. For both enzymes, a control group received the same treatment as the enzyme-digested material with the exception that no enzyme was added. SDS was then added to all samples to a final concentration of 0.2% and the samples were applied to a Sepharose CL-6B column (1 X 80 cm) eluted with 0.2% SDS in buffer B. Fractions of 1.5 ml were collected and assayed for radioactivity by scintillation counting. Nitrous acid deamination To determine the heparan sulfate content of the gradient fractions, nitrous acid deamination was used as described previously (18). The gradient peaks were divided into experimental (treated with 1.8% sodium nitrite in 1.8 M acetic acid) and control (treated with acetic acid alone) halves and incubated for 80 min at room temperature. The reaction was terminated by addition of 2 M sodium sulfamate. All samples were brought to a final concentration of 0.2% SDS and chromatographed on a Sepharose CL-6B column eluted with 0.2% SDS in buffer B. Mate-rials. The following materials were purchased: [6-gi[lglucosamine hydrochloride and NazsSO, from Amersham Corporation; Pronase and Streptomyces hyaluronidase from Calbiochem Corporation; testicular hyaluronidase from Worthington Biochemical Corporation; cesium chloride and guanidine hydrochloride from Bethesda Research Laboratories; Sepharose CLdB and CL-6B from Pharmacia Fine Chemicals, Inc.; EGTA from Eastman Organic Chemicals; chondroitinases ABC and AC from Miles Laboratories, Inc.; calf serum from K. C. Biological, Inc.; plastic tissue culture dishes from Lux Scientific Company, reference standard GAGS kindly provided by Drs. Cifonelli and Mathews of the University of Chicago. RESULTS
GAG composition and extractability. The GAG distribution of the substratum attached material from KiMSV cells was first characterized by the methods of Rollins and Culp (18) and compared to the distribution in the adhesion sites of Balb/c 3T3 cells from which these transformed cells were derived (Table I). L-SAM and R-SAM
MIKETO
450 TABLE
I
GLYCOSAMINOGLYCANDISTRIBUTIONS Polysaccharide class*
Cell associated (%)
L-SAM (%)
R-SAM (%I
KiMSV
HS HA C6S C4S cos DS Total
72.0 10.9 ND” 5.7 1.8 9.6 100.0
39.0 24.3 5.2 15.2 6.3 10.0 100.0
25.6 20.8 10.7 18.2 8.2 16.5 196.0
Balb/c 3T3d
HS HA C6S c4s cos DS Total
48.8 17.6 0.8 5.7 3.1 24.0 106.66
26.3 22.7 2.3 22.4 23.6 2.7 100.0
80.2 4.5 1.6 8.0 4.8 0.9 loo.0
Cell line
’ Cells were radiolabeled for 72 h in medium containing [SHlglucosamine after which L-SAM and RSAM were prepared (Materials and Methods) and quantitatively solubilized with SDS. Specific GAG classes were identified as described by Rollins and Culp (18). These data are representative of three separate determinations. * HS, Heparan sulfate; HA, hyaluronic acid, C-6-S, chondroitin g-sulfate; C-4-S, chondroitin 4-sulfate; COS, unsulfated chondroitin; DS, dermatan sulfate. The standard error in measurement of these numbers for separate sets of samples varied from +6% for the high-concentration GAGS to +13% for the low-concentrated GAGS. ‘None detected. d These data are taken from Ref. (18) for comparison with the data of the KiMSV cells being analyzed in this study.
from KiMSV cells have similar distributions of the various GAG components. Both adhesion sites are depleted in the amount of heparan sulfate and are enriched for hyaluronic acid and the chondroitin sulfate classes when compared to the cell-associated material (that is, EGTA-detached cells). Substratum-attached material from long-term growing or reattaching Balb/c 3T3 cells differ, however, in their GAG composition, with the R-SAM containing much more heparan sulfate and less hyaluronic acid and chondroitin sulfate than
AND CULP
the L-SAM. The GAG compositions of the L-SAMs from both cells are similar, whereas the R-SAMs differ significantly. The much higher amounts of hyaluronic acid and chondroitin sulfates of KiMSV RSAM sites correlates with the indication (6) that hyaluronic acid and/or the chondroitin sulfates are involved in detachment of cells from the substratum; newly attaching KiMSV cells are much easier to detach during EGTA treatment than newly attaching 3T3 cells (data not shown). In order to study the reassociation patterns of the GAGS and PG’s in the adhesion sites, the substratum-attached material was extracted with 4 M guanidine hydrochloride (GuHCl) under conditions that do not irreversibly denature proteoglycan species in order to further study their potential to reassociate into highmolecular-weight complexes (9). The percentages of the adhesion site protein and polysaccharide extracted with 4 M GuHCl are presented in Table II for comparison with SDS-solubilized material, which quantitatively removes L-SAM or R-SAM (13). GuHCl, 4 M, extracted equal amounts of KiMSV L-SAM radiolabeled with [3H]glucosamine or ?SO,2-; however, significantly more ?SO$--labeled material was extracted from the reattaching adhesion sites than [3H]glucosamine-labeled material. In comparison to Balb/c 3T3 cells, KiMSV adhesion sites were more labile to the 4 M GuHCl treatment. Various properties of the KiMSV GAG-containing moieties were then examined by gel filtration chromatography or isopycnic density gradient analyses. Chrmatography of extracts under associative or dissociative conclitiona Substratum-attached material (long-term generated and extracted with 4 M GuHCl) was dialyzed against 0.4 M GuHCl to permit reassociation of molecules into complexes or against 0.2% SDS to completely dissociate aggregates (13). Chromatography of the extracts on Sepharose CL-2B columns eluted with the same buffers is shown in Fig. 1. None of the sulfated GAG appeared at the void volume of the column eluted with 4 M GuHCl (Fig. 1A) or with SDS (Fig. 1C) indicating that high-molecular-weight
SUBSTRATUM
ADHESION
OF TABLE
GuHCl
TRANSFORMED II
EXTRACTABILI~ Radioactivity
Cell type
Cell fraction
KiMSV
Balb/c
3T3
451
CELLS
[SH]Glucosamine
(% of total)* N&?!30,
L-SAM R-SAM
54 35
53 63
L-SAM R-SAM
46 21
NDc ND
a L-SAM or R-SAM radiolabeled with [*Hlglucosamine of Naza?SO1 were prepared as described under Materials and Methods. The dishes bearing adhesion sites were then extracted with 4 M GuHCl as described under Materials and Methods. An aliquot of the GuHCl extract was assayed for radioactivity and compared with an equal aliquot of an SDS extract of a parallel set of dishes (SDS quantitatively solubilizes material from adhesion sites); corrections were made in all samples for quenching. * Calculated by: radioactivity solubilized with GuHCl f radioactivity solubilized with SDS X 166. ‘Not determined. A
57
4M GuHCl
II
,,-I
I IO
1 1
09 OS
IO
20 30 40 FRACTION NUMBER
50
60
FIG. 1. Gel filtration chromatography of L-SAM under associative and dissociative conditions. PHIGlucosamineor =SOg--radiolabeled KiMSV L-SAMs were isolated as described under Materials and Meth-
complexes were not present under these dissociative conditions. However, significant sulfated GAG appeared at V, after dialysis aginst 0.4 M GuHCl (Fig. lB), demonstrating the formation of such complexes. This was further shown by pooling the VO-eluting material in Fig. lB, dialyzing it against SDS, and rechromatographing on an SDS-eluted Sepharose CL-2B column; all of the material shifted well into the included region of the column (K,, = 0.5-0.7). Furthermore, the 4 M GuHCl extract consistently showed intermediatesized sulfated complexes (fractions 20-30 of Fig. 1A) which were not present in SDS extracts (Fig. 1C). A sizable amount of gluods. A portion of the L-SAM extracted in 4 M GuHCl in buffer A was directly chromatographed on a Sepharose CL-2B column eluted with 4 M GuHCl in buffer A (A). The remaining L-SAM was brought to associative conditions by extensive dialysis against 0.4 M GuHCl in buffer A and a sample chromatographed on a Sepharose CL-2B column eluted with 0.4 M GuHCl in buffer A (B). A final sample of L-SAM was further dialyzed against buffer B to remove all guanidine hydrochloride and brought to dissociative conditions by extensive dialysis against 0.2% SDS in buffer B (C). Fractions were analyzed for eH (0) or as (0) radioactivity as described under Materials and Methods. Void and inclusion volumes were determined using blue dextran and dinitrophenylated glycine. Only the ordinate on the right in A is cpm ?S X 103, whereas the ordinates in B and C are cpm (without any multiplication factor).
452
MIKETO
AND
cosamine-radiolabeled (nonsulfated) material persisted at V, of Fig. 1C in SDS and was shown to be hyaluronic acid by its sensitivity to Streptmgces hyaluronidase (data not shown). These experiments indicate that a portion of the sulfated GAG extracts from L-SAM can reaggregate into large complexes which are partially sensitive to 4 M GuHCl and completely sensitive to SDS. Similar analyses were made of R-SAM extracts (Fig. 2). There was a significant amount of sulfated GAG at the void volume of the column eluted with 0.4 M GuHCl (Fig. 2B) that was not apparent in 4 M GuHCl extracts (Fig. 2A), similar to the situation with L-SAM in Fig. 1. On the other hand, the R-SAM extracts (4 M GuHCl) contain very little intermediatesized aggregates (compare fractions 20-30 50
A.
4M
1000 1
GuHCl
4.5
900
2.5 _
em 700 - 600 500
2.0 -
- 400
4.0 "0 i
3.5 . 3.0 -
Vi 4
1.5 .
xx)
1.0 5F 0.5
200 100
M f 4.5 6. 7
0.4M
:: E
GuHCl
;; 4.0 4 3.5
800
3.0
600;
In, * z _o ” ,I
F6
‘;
7007 8
2.5
500
,,*
2.0
400
1.5
3cxJ
; c)
1.0
200
0.5
loo
FRACTION
NUMBER
FIG. 2. Gel filtration chromatography of R-SAM under associative and dissociative conditions. [“HIGlucosamineor ?S@--radiolabeled R-SAM samples were treated and analyzed as described in the legend to Fig. 1 for chromatography on columns of Sepharose CL-2B.
CULP
A.
ASSOCIATIVE -50 . 4.5
25.
1,.
I.0 20
. .-. 3.0 40 5D 61) 70 8.0 9D
IO0
I
FIG. 3. Isopycnic density gradient centrifugation of L-SAM. L-SAM radiolabeled with ~lglucosamine (O l ), or Naaa6S0, (0 - - - 0) was centrifuged on CsCl gradients under associative (A) or dissociative conditions (B) as described under Materials and Methods. Associative L-SAM was prepared by dialyzing L-SAM harvested in 4 M GuHCl against 0.4 M GuHCl in buffer A for ‘72 h. L-SAM extracted in 4 M GuHCl was directly applied to the CsCl gradient for centrifugation under dissociative conditions. Fractions of 0.5 ml were collected from the bottom of gradient tubes and assayed for ‘H (O-O), “S (0 - - - 0), or density (-) as described under Materials and Methods.
of Fig. 2A with Fig. 1A). GuHCl, 4 M, extracts of R-SAM also contained much less hyaluronic acid at the V, region of Figs. 2A or C when compared to L-SAM extracts. Density gradient centr&gaticm of extracts mder associative w dissociative umditims. When long-term generated substratum-attached material was centrifuged to equilibrium in a CsCl gradient under associative conditions, three peaks of radiolabeled material were observed (Fig. 3A)-GJ, GA2, and GA3 at densities of 1.67-1.71, 1.62-1.65, and 1.6-1.62 g/cm3, respectively. Although all three compo-
SUBSTRATUM
ADHESION
nents contain [3HJglucosamine-radiolabeled material, only GA1 and GA3 contained sulfated GAG. Similar peaks were observed with the associative extracts of Balb/c 3T3 L-SAM by Garner and Culp (13). Under dissociative conditions, KiMSV extracts generated a sizable pool of material at the top of the gradient (Go2 in Fig. 3B) and a small amount of sulfated GAG at the bottom of the gradient (Gnl). Similar analyses of R-SAM extracts on density gradients are shown in Fig. 4, and there are significant differences with the L-SAM profiles of Fig. 3. There is virtually no GA2 material in R-SAM centrifuged under associative conditions (Fig. 4A) and much less G,3; most of the material is highly sulfated and dense, appearing in the bottom fractions of the gradient in GAl. Another difference is noted when R-SAM is analyzed under dissociative conditions (Fig. 4B)-much of the sulfated material remains highly dense and does not shift to the top of the gradient as observed in L-SAM (Fig. 3B). Although analyses for specific GAGS demonstrate very similar distributions from both L-SAM and RA
B
ASSOCIATIVE
DISSOCIATIVE
1.0 20 30 40 50 60 70 80 VOLUME (ml)
90 10.0
FIG. 4. Isopycnic density gradient centrifugation of R-SAM [8H]Glucosamine- or Naz%@--radiolabeled R-SAM samples were prepared and analyzed on CsCl associative or dissociative gradients as described in the legend to Fig. 3.
OF TRANSFORMED 1600
I400
A. L-SAM (GlcN)
453
CELLS
Gnl
1200 5 1000 5L? so0 L 600 \ E 400 8 -?Q 200 x
IO
20
30
FRACTION
40
50
60
70
NUMBER
FIG. 5. Sensitivity of aminosugar-radiolabeled GJ to hyaluronidase digestion. IjHlglucosamine-radiolabeled L-SAM or R-SAM were extracted with 4 M GuHCl, dialyzed against 0.4 M GuHCl, and centrifuged on an associative cesium chloride gradient (Fig. 3A or 4A) as described under Materials and Methods. Fractions from the GJ peaks were pooled and treated with testicular hyaluronidase (A A), Streptomyces hyaluronidase (A---A) or no enzyme as a control (O 0). After enzymatic digestions, samples were chromatographed on a Sepharose CL-6B column eluted with SDS buffer as described under Materials and Methods. (A) PH]Glucosamine L-SAM G*l; (B) [8Hlglucosamine R-SAM GJ.
SAM (Table I), the 4 M GuHCl extracts of these adhesive materials behave quite differently upon density gradient analyses. Biochemical characterization of spe&$c GAG’s in density gradient peaks. We then chose to identify specific GAG moieties in the density gradient fractions of L-SAM and R-SAM. Because of the paucity of material, we were forced to use sensitivity to specific GAG hydrolases or chemical agents, along with shifts in material upon gel filtration chromatography, as assays for specific GAG classes. A comparison of the enzymatic sensitivities of associative extract GA1 pools of L-SAM or R-SAM is displayed in Fig. 5 for aminosugar-radiolabeled material. The control digest of L-SAM (Fig. 5A) generates two well-separated entities upon chromatography on Sepharose CLGB in an SDS-containing buffer-one minor moiety
454
MIKETO
AND
eluting just after V. and a major component with K,, = 0.45-0.55. The minor moiety is completely sensitive to Strepb myces hyaluronidase, demonstrating that this is HA, and some of the major component is also sensitive to the Streptomyces enzyme, although more of this material is sensitive to testicular hyaluronidase (chondroitin sulfate-containing moieties). R-SAM GA1 is considerably different (Fig. 5B), with virtually no HA (Streptomyces hyaluronidase insensitivity) but comparable amounts of chondroitin sulfate to L-SAM GAl. Treatment of sulfate-radiolabeled material from GA1 with either testicular hyaluronidase or nitrous acid can distinguish heparan sulfate from chondroitin sulfate entities (Fig. 6). From L-SAM, approximately 30% of the major sulfated peak is sensitive to the testicular enzyme (CS)
20
30 FRACTION
40
60
60
7'0
NUMBER
FIG. 6. Sensitivity of ‘%Oz--radiolabeled G*l to various treatments. L-SAM and R-SAM from “SO:--radiolabeled KiMSV cells were extracted with 4 M GuHCl, dialyzed against 0.4 M GuHCl, and centrifuged on an associative cesium chloride gradient (Figs. 3A and 4A) as described under Materials and Methods. Fractions containing GJ were pooled and split into three samples. The first sample was digested with testicular hyaluronidase (A A), while the second sample was treated with nitrous acid (0 - - - 0). The third sample was left untreated as a control (0-O). All three samples were chromatographed on a Sepharose CL-6B column eluted with an SDS buffer as described under Materials and Methods. (A) &SO:--radiolabeled L-SAM GAl; (B) “SO:--radiolabeled R-SAM Gnl.
CULP
4.or
‘A2
1
3.5 p 3.0 Y g 25E " 2.0 72 ; 1.5Y " & l.On 0.51
IO
20
30
FRACTION
40
50
60
NUMBER
FIG. 7. Sensitivity of GA2 to Streptcrm~ces hyaluronidase digestion. pH]Glucosamine-radiolabeled L-SAM was centrifuged under associative conditions as described under Materials and Methods and Fig. 3A. Fractions from peak GA2 were pooled and split into two parts after dialysis against digestion buffers. One sample was treated with Streptom~ces hyaluronidase (0 - - - 0) while the other sample was left untreated as a control (0 0). Both samples were chromatographed on a Sepharose CL-6B column eluted with SDS-containing buffer.
while the remaining 70% is sensitive to breakdown with nitrous acid (HS). Similar sensitivities were observed for R-SAM GA1 (Fig. 6B), with the single exception that a higher proportion of the major peak was HONO-sensitive. The results of Fig. 5 and 6 indicate that the dense material banding at the bottom of associative gradients is principally HS proteoglycan, with a smaller amount of CS proteoglycan and, in the case of L-SAM, some hyaluronic acid. Since HA alone bands at much lighter densities in these gradients (see below), this “dense” HA must be complexed with one or more of the proteoglycan species. GA2 from L-SAM (Fig. 3A) is entirely HA, as determined by its complete sensitivity to Streptomgces hyaluronidase (Fig. 7). Similarly, when nonradiolabeled HA was analyzed under identical conditions, it bands only in the GA2 region of gradients and not in GA1 or GA3 regions (data not shown). Therefore, there is a large excess of HA in the 4 M GuHCl extracts of L-SAM (and no GA2-containing HA in the 4 M GuHCl extract of R-SAM) and this moiety should not be a limiting factor in
SUBSTRATUM
ADHESION
OF
formation of supramolecular aggregates of these extracts (9). The content of GAG in GA3 was then determined. Sensitivity to enzyme digestion of aminosugar-radiolabeled material is shown in Fig. 8. The control digest of LSAM generates a large peak at Vi and a smaller included peak when eluted from Sepharose CL-6B columns in an SDS-containing buffer (Fig. SA). The Vi material is completely sensitive to Streptomvces hyaluronidase showing that this is HA. This could be some cross-contaminating HA from the GA2 peak (see Fig. 3A) or a separate class of HA. Interestingly, R-SAM also contained this class of HA in GA3 (Fig. SB), although R-SAM ccmtains no GA.2peak; 2.2 2.0
I.6 f
A
L-SAM (GlcN)
GA3
I.0 g
0.6
2
0.6
E
0.4
ho.2 0 P-l E2 x z g
I.6
g
1.2
14
IO
20
FRACTION
30
40
50
60
NUMBER
FIG. 8. Sensitivity of GA3 to hyaluronidase digestion. GA3 fractions (see Figs. 3A and 4A) of mlucosamine-radiolabeled L-SAM or R-SAM were pooled separately, dialyzed against digestion buffers, and digested with testicular hyaluronidase (A A) or Streptomgca hyaluronidase (A - - - A). After enzymatic digestions, samples were chromatographed on Sepharose CL-6B columns eluted with SDS-containing buffers for comparison with elution patterns of an undigested control (0-O). (A) [4I]Glucosamine-radiolabeled L-SAM Gn3; (B) [SHlglucosamine-radiolabeled R-SAM Gn3.
TRANSFORMED
CELLS
5
16-
A. L-SAM (GlcN)
I= 2 &
14. 12.
V, \
g
IO-
455
G,l
0.6 -
FIG. 9. Sensitivity of L-SAM Go1 to various treatments. Gnl from L-SAM extracts after dissociative analysis on a cesium chloride gradient (Fig. 3B) was pooled, dialyzed against the appropriate buffer, and treated with either testicular hyaluronidase (A A), nitrous acid (0 -. - 0), or left untreated (O 0). Samples were then chromatographed on a Sepharose CL-6B column in an SDS-containing buffer as described under Materials and Methods. (A) [SHlglucosamine-radiolabeled L-SAM Gnl; (B) %O:--radiolabeled L-SAM Gnl.
it appears likely then that the HA in GA3 is a separate class of HA with possibly glycoprotein bound to it to give it a much lighter density. A very small amount of the included material of L-SAM GA3 (Fig. SA) was sensitive to testicular but not Streptomyces hyaluronidase, indicating some CS. This was not the case for R-SAM GA3. All of the sulfate-radiolabeled GA3 from L-SAM was sensitive to testicular hyaluronidase but not to HONO, demonstrating again some CS in GA3 but no HS (data not shown). It appears that most of the included aminosugar-radiolabeled material in GA3 is glycoprotein. The small amount of Gnl from L-SAM dissociative gradients which persists at high density was tested for its sensitivity (Fig. 9). The high-molecular-weight aminosugar-radiolabeled material at VO is sensitive to testicular hyaluronidase (Fig. 9A) but not to Streptmyces hyaluronidase and therefore must be proteoglycan-containing CS sequences (Pronase digestion shifts this material into included regions
456
MIKETO
of the profile) (data not shown). Approximately one-half of the sulfate-radiolabeled Gnl is CS (sensitive to testicular hyaluronidase) while the remainder is HS (sensitive to HONO) (Fig. 9B). R-SAM Gnl content is considerably different (Fig. 10). The vast majority of the sulfate- or aminosugar-(not shown) radiolabeled material is HS with only a minor fraction of CS. DISCUSSION
These results have characterized in a preliminary fashion some properties of proteoglycans from the newly formed or “matured” substratum adhesion sites of a metastatic malignant derivative of Balb/ c. 3T3 cells, KiMSV (3, 14). Considerable evidence is accumulating that heparan sulfate proteoglycans play a direct role in forming substratum adhesion sites whereas hyaluronic acid may function in the detachment of cells (6, 7, 15, 16, 19). The ability of both cellular and plasma fibronectins to bind to HS sequences while only the aggregated form of cellular fibronectin binds to HA is consistent with these proposals (1’7). It, therefore, becomes imGDl
R-SAM
9 ]b
(SO&')
Es o 0.8 5x 0.6 'I* 8 0.4 x 0.2 IO
20
FRACTION
30
40
50
60
NUMBER
FIG. 10. Sensitivity of R-SAM Gnl to testicular hyaluronidase or nitrous acid. Gnl fractions from %3@--radiolabeled R-SAM extracts were pooled, dialyzed against the appropriate buffer, and split into three samples. One sample was digested with testicular hyaluronidase (A A), the second treated with nitrous acid (0 -a - 0), and the third left untreated as a control (0 0). All three samples were chromatographed on a Sepharose CL-6B column eluted with an SDS-containing buffer as described under Materials and Methods.
AND
CULP
portant to biochemically characterize the hyaluronate and various GAG-containing proteoglycans from the substratum-attached material of these cells in order to better understand their potential functions. The data reported herein demonstrate some significant differences in the GAG moieties from the adhesion sites of KiMSV cells when compared to the parental Balb/c 3T3 cells (13). In particular, the RSAM of KiMSV cells contains much more HA and CS than the R-SAM of 3T3 cells; this may reflect the considerable ease of detachment of KiMSV cells from the substratum under all culture conditions. However, much of the HA in KiMSV RSAM is nonextractable with 4 M GuHCl but becomes extractable with “maturing” of these sites into L-SAM. Furthermore, the GA3 density gradient pool of KiMSV cells contained considerable “light density” HA but no HS; Balb/c 3T3 GA3 contained very little HA but significant amounts of HS (13). A much smaller proportion of proteoglycan material extractable with 4 M GuHCl from KiMSV L-SAM or R-SAM could reaggregate when dialyzed against associative buffers (as analyzed by gel filtration chromatography) when compared to 3T3 cells (13). On the other hand, KiMSV extracts in either 4 M or 0.4 M GuHCl contained intermediatesized aggregates sensitive to SDS; 3T3 extracts generally contained only high-molecular-weight aggregates excluded from Sepharose CLZB columns (13). All of these data indicate appreciable quantitative and/or qualitative differences in the proteoglycan moieties from the adhesive material of these two cell types that display very different growth potentials in animals and significantly different adhesive behaviors on tissue culture substrata. In agreement with the previous data of Garner and Culp (13) with Balb/c 3T3 cells, there are differences in the 4 M GuHCl extracts of newly formed adhesion sites (RSAM) or “matured” sites (L-SAM) of KiMSV cells. R-SAM contains much less GuHCl-extractable HA than L-SAM as demonstrated with the I’,,-eluting material from gel filtration columns (Figs. 1 and 2) or the GA2 band of isopycnic density
SUBSTRATUM
ADHESION
gradients (Figs. 3 and 4). The presence of HA in GJ of L-SAM associative gradients is reminiscent of the HA:chondroitin sulfate proteoglycan aggregates found in cartilage (9); similar aggregates have been found in the reassociated extracts of tissue cultured human fibroblasts (11, 12). Whether the HA in this high-density material from L-SAM (but not R-SAM) is truly bound to CS-PG remains to be proven. Although R-SAM contains no intermediate-density GA2, whereas L-SAM contains much of this material, R-SAM contains considerable “light density” HA in a smaller total pool of GA3. L-SAM contained a small amount of HA in GA3, which could possibly be explained by cross-contamination by the large amount of HA in L-SAM GA2. The nature of the material which binds to HA under these associative conditions to make it “light” is of considerable interest. Another important difference between R-SAM and L-SAM extracts is the behavior of heparan sulfate moieties. The vast majority of the HS in L-SAM shifts to the top of dissociative gradients, whereas most of the HS in R-SAM remains in the high density fractions of dissociative gradients. One explanation for this derives from the data of Garner and Culp (13), namely, that GD1 HS occurs as large proteoglycans, whereas L-SAM Go3 HS occurs as free HS chains. This would indicate that newly formed adhesion sites contain principally HS-PG that is later catabolized by some unknown mechanism into free HS chains. Alternatively, the light-density HS may be in the form of a proteoglycan with a hydrophobic region of the core protein which allows these to aggregate into lowdensity complexes (20); R-SAM and L-SAM would then have very different distributions of such a membrane-associated proteoglycan and there would be metabolic conversion of one form of the proteoglycan into the other form. Clearly, more information will be required to prove or disprove such a metabolic conversion process in the adhesion sites of cells. This would raise the important issue as to whether metastatic and invasive cell types have a greater capacity to catabolize the proteoglycans in their substratum adhesion sites
OF
TRANSFORMED
CELLS
457
when compared to their normal cell counterparts, an issue that will be directly addressed in future studies. ACKNOWLEDGMENTS This work was supported by a National Institutes of Health Research Grant AM25646. The authors wish to thank Dr. Judy Garner and Michael Lark for many helpful discussions. REFERENCES 1. CULP, L. A. (1978) in Current Topics in Membranes and Transport (Juliano, R. L., and Rothstein, A., eds.), Vol. 11, pp. 327-396, Academic Press, New York. 2. POSTE, G., AND FIDLER, I. J. (1980) Nature (London) 283, 139-146. 3. STEPHENSON, J. R., AND AARONSON, S. A. (1972) J. Exp. Mea! 135.503-514. 4. C~LP, L. A., MURRAY, B., AND ROLLINS, B. J. (1979) J. Supram Struck. 11.401-427. 5. ROSEN, J. J., AND CULP, L. A. (1977) Exp. Cell Res 107, 139-149. 6. ROLLINS, B. J., CATHCART, M. K., AND CLJLP, L. A. (1982) in The Glycoconjugates (Horowitz, M. I., ed.), Vol. III, pp. 289-329, Academic Press, New York. 7. LATERRA, J., SILBERT, J. K., AND CULP, L. A, (1983) J. Cell Biol 96, in press. 8. ALTENBURG, B. C., SOMERS, K., AND STEINER, S. (1976) Cancer Res. 36,251-260. 9. HASCALL, V. C. (1977) J. Szqraml Struet. 7, lOl120. 10. ROLLINS, B. J., AND CIJLP, L. A. (1979) Biodwmistry 18.5621-5629. 11. COSTER, L., CARLSTEDT, I., AND MALMSTROM, A. (1979) Biochem J. 183.669-677. 12. VOGEL, K. G., AND PETERSON, D. W. (1981) J. Bid Ch 256,13235-13242. 13. GARNER, J. A., AND CULP, L. A. (1981) Biochxmistry 20, 7350-7359. 14. NICOLSON, G., BRIJNSON, K. W. B., AND FIDLER, I. J. (1978) Cancer Res. 38.4105-4111. 15. BARNHART, B. J., Cox, S. H., AND KRAEMER, P. M. (1979) Exp. Cell Rex 119, 327-332. 16. RICH, A. M., PEARLSTEIN, E., WEISSMANN, G., AND HOFFSTEIN, S. T. (1981) Nature (London) 293, 224-226. 17. LATERRA, J., AND C~LP, L. A. (1982) J. Bid Ch 257,719-726. 18. ROLLINS, B. J., AND C~LP, L. A. (1979) Biochanistry 18.141-148. 19. KNOX, P., AND WELLS, P. (1979) J. CeU Sci 40,7789. 20. KJELLEN, L., PETTERSSON, I., AND HOOK, M. (1981) Proa Nat. Acad Sci USA 78, 5371-5375.