[CANCER RESEARCH 45, 2632-2641, June 1985]

Relationship between the Organization and Synthesis of Vimentin and theMetastatic Capability of B16 Melanoma Cells1

Avri Ben-Ze'ev2 and Avraham Raz3

Department of Genetics [A. B-Z.] and Cell Biology [A. R.¡,The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT

The organization and synthesis of the vimentin-containingcytoskeletal network as well as the metastatic capability of B16-F1 melanoma cells were investigated in cells treated with cyclo-heximide (CH). The addition of CH to cells for 4-8 h resulted ina marked reversible alteration in the organization of the vimentin-containing network in B16-F1 melanoma cells as well as in a

variety of epithelial and fibroblast cell lines. Treatment of cellswith CH led to a reduction in the synthesis of vimentin, tubulin,and actin followed by a decrease in the concentrations of mRNAscoding for these proteins. However, out of these three cytoskeletal elements, only the organization of the intermediate filamentswas disrupted by CH. Cells treated previously with CH andinjected i.v. into syngeneic mice had diminished capacity to formlung métastases as compared to control untreated cells. Thiseffect was reversible, and the metastatic capability recovered tothe control level after the drug was removed from the growthmedium for 16 h. The possibility that the organization and thesynthesis of the cytoskeletal components are related and thatthe metastatic capability of B16 melanoma is influenced by theorganization of the cytoskeletal networks are discussed.

INTRODUCTION

It is now generally accepted that cell geometry can play acentral role in the control of cell proliferation (15), and that manyother growth-related cellular activities are probably also mediated

through changes in cell shape (3, 9). Our studies on cell configuration-related gene expression led us to conclude that changesin cell shape bring about responses at the level of macromolec-

ular metabolism (i.e., DNA, RNA, and protein synthesis) (1, 7, 8,13). The suggestion that the progressive loss of these cell shape-

responsive metabolic controls in transformed mouse fibroblastsis related to the phenomenon of tumor progression (33) and thatthe expression of the neoplastic phenotype in C3H/10T1/2 cellscould be directed by changes in cell shape (10) has promotedthe search for the possible role of cell shape in the expressionof the malignant phenotype. Recently, we have analyzed theresponse of tumor cell variants exhibiting distinct metastaticcapability to changes in cell shape (25) and demonstrated areversible modulation in the metastatic lung colonizing potentialof B16 melanoma by varying cell configuration (26). Variations incell shape that bring about alterations in the metastatic capabilityare also accompanied by a marked decrease in the synthesis of

1Supported by a grant from the National Council for Research and Development,

Israel, and the German Cancer Research Center, Heidelberg, West Germany.2 Incumbent of the Carl and Frances Kom Career Development chair in perpe

tuity.3 Incumbent of the Sophie M. T. and Richard S. Richards Career Development

chair in cancer research in perpetuity.Received 9/5/84; revised 12/26/84; accepted 2/6/85.

the intermediate filament protein, vimentin (2,26). Since elementsof the cytoskeleton are thought to have a role mainly in determining cell shape and are suggested to be involved in controllingcytodynamic processes related to tumor cell dissemination andmetastasis (17, 18, 21), we have studied the expression ofcytoskeletal components in relation to changes in cell shape,adhesion, motility, and malignancy (8, 21, 24-27, 32). In the

present study, we examined the response of cytoskeletal proteingene expression to the alteration of the intermediate filamentnetwork by CH,4 a potent inhibitor of protein synthesis that was

shown to disrupt the vimentin network in CV-1 cells (28). Inaddition, this alteration in the vimentin-containing network ena

bled us to investigate the possible linkage between the organization of the vimentin network and the lung-colonizing ability ofB16-F1 melanoma cells.

MATERIALS AND METHODS

Cells and Culture Conditions. MDBK, CV1, and BSC-1 green monkey

kidney epithelial cells were from the American Type Culture Collection(Rockville, MD). The Swiss 3T3 mouse fibroblasts were obtained fromDr. H. Green (Harvard Medical School, Boston, MA). The human foreskinfibroblasts were obtained from Dr. M. Revel (The Weizmann Institute ofScience, Rehovot, Israel), and the B16-F1 mouse melanoma was from

Dr. I. J. Fidler (M. D. Anderson Hospital and Tumor Institute, Houston,TX). All cell lines were grown as monolayers on plastic in Dulbecco'smodified Eagle's medium (Grand Island Biological Co.) containing 10%

heat-inactivated fetal bovine serum (Bio-Lab), nonessential amino acids,and antibiotics. The cells were maintained at 37°C in a humidified

atmosphere of 7% CO2-93% air. To ensure reproducibility of in vivo and

in vitro assays, all experiments were done with cultures grown for nolonger than 4 weeks after recovery from frozen stocks. The cells wereharvested from cultures in their exponential growth phase by washingthe cell monolayer with warm Ca2+- and Mg2*-free PBS and overlaying

the cells with a solution of 2 rrtM EDTA in the same buffer for 3 min at37°C. The cells were then washed and resuspended in PBS. Only cell

suspensions exhibiting >95% cell viability (measured from the proportionof cells excluding trypan blue) and free of aggregates were used in thesestudies. The cells were treated with CH, 50 Mg/ml, or with ara-C, 50 ^g/

ml, by adding the inhibitors directly to the medium from a 100x solutionin H2O. The cells were labeled with L-^SJmethionine at 100 /¿Ci/ml(1250Ci/mmol; Amersham, England) for 60 min in methionine-deficient medium

containing 10% dialyzed fetal bovine serum.Experimental Pulmonary Métastases. Unanesthetized 8-week-old

female C57BL/6 mice (The Jackson Laboratory) were inoculated i.v. inthe tail vein with 5 x 104 tumor cells in 0.2 ml of PBS. After 18 days, the

mice were autopsied, and their lungs were removed, rinsed, and fixedwith 5% formaldehyde in PBS. The number of tumor colonies in thelungs was determined by counting under a dissecting microscope (Zeiss).

Cell Fractionation. A Triton X-100 Insoluble cytoskeletal fraction

enriched in intermediate filaments was prepared in a buffer containing

4The abbreviations used are: CH, cycloheximide; ara-C, 1-0-o-arabinofurano-sylcytosine; poly(A)1, polyadenylated; MDBK, Madin-Darby bovine epithelial cells;

PBS, phosphate-buffered saline, pH 7.2; SDS, sodium dodecyl sulfate.

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0.3 M sucrose, 10 mw Tris-HCI (pH 7.4), 50 rriM NaCI, 2.5 HIM MgCI2,0.5% Triton X-100, and 1 mw phenylmethylsulfonyl fluoride as describedbefore (6). The proteins in the Triton X-100 soluble fraction were con

centrated by ethanol precipitation, and the samples were solubilized insample buffer for gel electrophoresis.

Protein Gel Electrophoresis. Proteins were analyzed by SDS (sodiumdodecyl sulfate) gel electrophoresis in 10% polyacrylamide slab gelsaccording to Laemmli (20) or by 2-dimensional gel electrophoresis according to O'Farrell (23).

RNA Extraction, in Vitro Translation, and RNA Blot Hybridization.Poly(A)+ RNA was isolated from the cytoplasm of cells and 1 ng ofpoly(A)+ RNA was used for in vitro translation in a reticulocyte cell-freelysate as described before (13). Poly(A)+ RNA, 5 ^g, was used for RNA

blot experiments, where the RNAs which were separated on agarosegels and were transferred to nitrocellulose paper (13) were hybridized todCTÅ“P-labeled nick-translated plasmid DNA containing vimentin (12),

actin, or tubulin sequences (16), as described before (13).Fluorescent Labeling of Cells. Cells grown on glass coverslips were

fixed and permeabilized by incubating for 20 min at -20°C in absolutemethanol and then for 5 min in absolute acetone at -20°C. The cells

were incubated at room temperature with anti-vimentin antibody (ob

tained from Dr. R. 0. Hynes, M. I. T., Cambridge, MA), and after extensivewashing in PBS, the cells were incubated with rhodamine-labeled goatanti-rabbit IgG (obtained from Dr. B. Geiger, Weizmann Institute of

Science, Rehovot, Israel). The fluorescently labeled cells were examinedwith a Zeiss photomicroscope III. For the visualization of actin-containingfilaments, the cells were fixed at 22°Cwith 3.5% formaldehyde in PBSand permeabilized by incubating at 22°Cfor 10 min with 0.5% Triton X-

100 in PBS followed by incubation for 30 min with rhodamine-phalloidin

(provided by W. W. Franke, German Cancer Research Center, Heidelberg, West Germany). Microtubules were visualized in cells that werefixed and permeabilized for 2 min in Hanks' buffer containing 0.5% Triton

X-100, 0.25% glutaraldehyde, 5 ITIMmorpholinoethanesulfonic acid (pH6.1), 2 nriMEGTA, and 2 HIM MgCI2 followed by a 10-min incubation in0.1% glutaraldehyde in the same buffer but without Triton X-100, andtwo 10-min successive incubations in NaBH3, 0.5 mg/ml, at 4°C.The

microtubules were stained with rabbit anti-tubulin antiserum (provided

by Dr. F. Solomon, M. I. T., Cambridge, MA) and rhodaminated goatanti-rabbit IgG, as described for vimentin filaments above.

RESULTS

CH-induced Alteration in the Vimentin-containing Network

Organization. In an attempt to reversibly modify the organizationand/or synthesis of the vimentin-containing network, we decided

to use CH since it was demonstrated (28) that CH induces aspecific disruption of vimentin filaments organization in monkeykidney CV-1 cells. Therefore, a variety of cell types was treatedwith CH for 6 to 8 h, and the organization of the vimentin-containing intermediate filament network was examined by im-munofluorescence (Figs. 1 to 3). Fig. 1, A' to C', shows an

alteration in the organization of the vimentin network in CV-1cells (Fig. 1-4'), Swiss 3T3 cells (Fig. 16'), and in the MDBKepithelial cell line (Fig. 1C ') as compared to the control untreatedcells (Fig. 1, A to C). Fig. 2A ' shows that the vimentin-containing

network of the monkey kidney BSC-1 cell line is similarly affected

by CH as compared to the control (Fig. 2A). The characteristicwavy filaments stained by the anti-vimentin antibody in untreated

cells (Figs. 1, A to C and 2/4) were no longer observed in CHtreated cells. Instead, the vimentin filaments appeared patchyand aggregated (Figs. 1, A' to C' and 2A'). In contrast, no

alterations were induced by CH in the organization of the microtubules (Fig. 2ß')or of the microfilaments (Fig. 2C ') as compared

to untreated BSC-1 cells (Fig. 2, B and C).

Since we have shown previously that changes in cell shape ofB16 melanoma cells bring about an inhibition of vimentin synthesis (2, 26), we have examined next the vimentin network in B16cells treated with CH (Fig. 3). In spite of the fact that these cellsare relatively poorly spread on a solid substrate, it was possibleto observe marked alterations in the intermediate filament network of these cells (Fig. ZA) after treatment with CH for 4 h (Fig.3B) or 8 h (Fig. 3C). When the drug was removed and the cellswere allowed to recover for 16 h, the vimentin network reorganized to its original pattern (Fig. 3D). This treatment with CH didnot lead to conspicuous changes in cell morphology (Fig. 3, A 'to D'), and the cells remained attached and spread on the solid

substrate.Inhibition of Vimentin Synthesis in Cells Recovering from

CH Treatment. We have demonstrated previously that changesin cell shape, or certain alterations in the organization of thevimentin-containing network (2, 4, 26) are followed by a specific

decrease in vimentin synthesis. Thus, it was of interest to examine the rate of vimentin synthesis in cells after treatment withCH. A variety of established cell lines was treated with CH for 6h, the drug was then removed, and the cells were extensivelywashed and then labeled for 1 h with pSJmethionine. A TritonX-100 insoluble cytoskeletal fraction enriched in vimentin filaments was prepared, and similar amounts of trichloroacetic acid-

precipitable radioactive protein samples were analyzed by SDSgel electrophoresis. Fig. 4 shows that in all cells tested therewas a marked reduction in the rate of vimentin labeling relativeto other proteins in CH-pretreated cells (Fig. 4, Lanes 2) as

compared to untreated cells (Fig. 4, Lanes 1), or as comparedto CH-treated cells that were allowed to recover for 16 h priorto pulse labeling with [^SJmethionine (Fig. 4, Lanes 3). While the

labeling of many other proteins appeared also to be affected bythe CH treatment (see also below), the effect on vimentin labelingappears to be most dramatic.

Since we were interested in analyzing the biological propertiesof the B16 melanoma cells that were treated with CH, thebiochemical studies presented below were performed mainlywith this cell line, but similar results were obtained with othercell lines. Fig. 5 shows that the decrease in vimentin labeling inB16 melanoma cells is apparent already after 2 h of CH treatment(Fig. 5, Lane 2) and that vimentin labeling is extensively reducedafter 8 to 11 h of treatment (Fig. 5, Lanes 5 and 6). Afterprolonged treatment with CH the synthesis of actin is alsoreduced. If the cells are allowed to recover for 8 h from CHtreatment, the level of vimentin synthesis recovers to controlrates (Fig. 5, Lane 8). Two-dimensional isoelectrofocusing SDSgel analysis (Fig. 5, A to C) of Triton X-100-insoluble cytoskel-

etons identified the position of the radioactive vimentin spot thatwas reversibly reduced in CH-treated cells.

Reduction in Vimentin mRNA Content in CH-treated Cells.In order to analyze whether the decrease in vimentin labeling inCH-treated cells is the result of a decrease in the amount ofvimentin mRNA, poly(A)+ cytoplasmic RNA was prepared from

control and CH-treated B16 cells. By translating similar amountsof poly(A)+ RNA from untreated (Fig. 6A) and CH-treated (Fig.

6B) cells in a cell-free reticulocyte lysate in vitro and analyzingthe proteins by 2-dimensional gel electrophoresis, the following

results were obtained. The translational activity of the mRNAscoding for most proteins was unchanged in CH-treated cells in

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comparison with control cells (compare Fig. 6, A with ß),but theactivity of mRNAs coding for several major proteins including thecytoskeletal proteins vimentin, actin, and tubulin (see arrowheads) was markedly reduced in CH-treated cells. The sameresults were obtained by the analysis of the Triton X-100 solubleextract from cells treated with CH and pulse labeled with [KS]-

methionine that showed a significant decrease in both a and ßtubulin synthesis, a slight reduction in actin synthesis, and adramatic reduction in a high-molecular-weight protein (Fig. 6, C

and D, large arrowhead). The decrease in the in vitro translationalactivity of the vimentin, tubulin, and actin mRNAs is the result ofa decrease in the mRNA content of these proteins as shown bythe RNA blot hybridization with dCT32P-labeled complementary

DNA clones for vimentin (Fig. 6£),tubulin (Fig. 6F), and actin(Fig. 6G). By analyzing similar amounts of poly(A)+ RNA from

cells treated for various periods of time with CH, it appears thatafter 4 to 8 h of treatment with CH there is a dramatic reductionin the amount of vimentin, tubulin, and actin mRNAs, but thattheir level increases again to near that of the control untreatedcells 8 h after removal of the drug.

Metastatic Capability of B16 Melanoma Cells Treated withCH. We have demonstrated recently that by growing B16 melanoma cells for a short period of time in suspension rather thanas a monolayer culture it is possible to modulate reversibly themetastatic capability of these cells (26). These changes in cultureconditions that were characterized by changes in cell shape andprobably by changes in the organization of the cytoskeleton alsoinduced a specific reduction in vimentin synthesis (2, 26). Therefore, we were interested in examining the lung-colonizing capa

bility of cells treated with CH in which both the organization andthe rate of synthesis of vimentin were altered. Cells treated withCH for 4 h or 8 h or cells treated for 8 h with CH and thenallowed to recover for 16 h without the drug were injected intothe tail vein of syngeneic mice, and the incidence of pulmonarymétastaseswas determined 18 days thereafter. Table 1 summarizes 2 independent experiments in which a decrease in themetastatic capability of cells is already observed in B16-F1

melanoma cells after 4 h of treatment with CH. After 8 h oftreatment with CH, there was a 15- to 20-fold decrease in the

Table 1Incidence of pulmonary métastasesin C57BL/6 mice inoculated i.v. with 5 x JO*

B16-F1cellsTumor

celltreatmentExperiment

1Control4

h with CH (50»ig/ml)8

h with CH (50 ^g/ml)8 h with CH (50 ng/ml),

16 hrecoveryExperiment

2Control4

h with CH (50 »ig/ml)8 h with CH (50 jig/ml)8 h with CH (50 Kg/ml),

16 h recovery8 h with ara-C (50ng/ml)8

h with ara-C (50 >ig/ml),16 hrecovery*Numbers in parentheses,No.

of pulmonarymétastases5,7,

12, 14, 18,18,20,22.22,27, 42

4,5,7,7,7,8,8, 11, 11,17,18,23

0,0,0, 1,1,1,2,3,5,6,77,14,18,19,27,33,44,465,9,11,11,

11,14, 18,19,19,20

3,4,6,6,8,9,9, 10, 11,150,0,0,0,0,1,1,1,1,3,4

15,16, 16,18,20,22,26,372,5,9,11,15,18,18,19,

20, 24, 2911,12,14,15,19,20,24,27range.Median19

(5-42)"

8(4-23)1

(0-7)19(7-46)13

(5-20)9

(3-15)1 (0-4)

18(15-37)18

(2-29)

15(11-27)

number of tumor lung nodules. This effect could be reversed ifthe cells were allowed to recover from the drug treatment. Byusing a different drug, such as ara-C, that inhibits DNA synthesis

but has no effect on cytoskeletal protein organization or synthesis (results not shown), we did not observe changes in themetastatic propensity of the cells. Therefore, the results suggesta specific role for CH in inducing parallel changes in both thevimentin-containing network organization and cytoskeletal pro

tein synthesis as well as in the metastatic potential of B16melanoma cells.

DISCUSSION

In the present study, we examined the interrelationship between disruption of the vimentin-containing intermediate filamentnetwork, the synthesis of vimentin, and of the lung-colonizingcapability of the malignant B16-F1 melanoma cells. The resultsindicate that the kinetics of the CH-induced changes in vimentin

synthesis and organization and recovery to the control level afterremoval of the drug were paralleled by a similarly reversiblealteration in the lung-colonizing ability of B16 melanoma cells

that were treated with CH. By screening a variety of establishedcell lines, we could demonstrate that the organization of thevimentin network in cell lines derived from both epithelia (MDBK,CV-1, BSC-1) and fibroblasts (3T3, human foreskin) were affected. In addition, CH-induced alterations in the vimentin net

work were also observed in tumorigenic cells such as B16melanoma that were derived from the neural crest as well as inthe nontumorigenic cells described above. Reorganization of theintermediate filament network by CH did not result in an apparenteffect on the organization of the microtubules or of the micro-filaments. However, in CH-treated cells, we observed an inhibi

tion of vimentin synthesis that resulted from a decrease in thecontent of vimentin mRNA in the cytoplasm.

A similar phenomenon of disorganization and inhibition ofvimentin synthesis was demonstrated recently in CV-1 and BSC-

1 cells at late times after infection with SV40 (4). The decreasein vimentin synthesis after SV40 infection as well as in CH-

treated cells was accompanied by a similar decrease in the levelof vimentin mRNA in the cytoplasm which excludes a translational control for vimentin synthesis in these systems. We haveshown previously that when BSC-1, 3T3, MDBK, or B16 melanoma cells were cultivated in suspension rather than in mono-

layer, there was a marked decrease in vimentin synthesis (2, 5,26). These results altogether thus suggest that changes in cellshape or in the organization of the vimentin-containing interme

diate filament network may induce a feedback mechanism thatfinally results in a decrease in the level of vimentin mRNA in thecytoplasm. In a previous study, we and others have demonstrated a similar feedback control mechanism for tubulin synthesis (6, 11). In cells where the microtubules were disrupted bycolchicine, there was an inhibition of tubulin synthesis due to adecrease in the level of tubulin mRNA. The inhibition in thesynthesis of tubulin, actin, and a few other proteins in CH-treated

cells, in addition to that of vimentin, is as yet unclear. However,the inhibition of tubulin synthesis in CH-treated cells could resultfrom the short half-life of tubulin mRNA, as suggested previously(6). This is based on the finding that in cells treated with actino-

mycin D, there is a similar decrease in the level of tubulin mRNA(6, 11). The reason for the decrease in actin synthesis in CH-

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treated cells is unclear. Changes in actin synthesis as a result ofchanges in cell shape were observed in 3T3 and 3T6 cells, wherewe have shown that actin mRNA expression changes dramatically during the cell configuration changes that accompany cellgrowth (1, 13). It appears therefore that the regulation of genescoding for all 3 major cytoskeletal proteins may be linked to themorphological organization of the cytoskeleton.

It was recently suggested that the progressive loss of cellconfiguration sensitive macromolecular metabolism control maybe related to the gradual independence from growth restrictionsacquired during tumor progression (33). Several reports suggested, however, that the expression of the malignant phenotypeor the metastatic capability can be modulated by changes in cellshape (10, 26) or in the adhesive properties of the cells, asshown in cells treated with dimethyl sulfoxide (30). Furthermore,the selection of cells that are adherent to plastic from a suspension culture of a highly malignant T-cell lymphoma revealed that

the adherent cells lost their metastatic capability (14). In anotherstudy with S49 lymphoma cells which were grown in suspension,it was demonstrated that adhering variants of this cell lineexpress impaired tumorigenicity (19). Cell rounding and reducedcellular adhesion induced by prolactin were also suggested toplay a role in determining the invasive and metastatic potentialof human breast cancer cells (29). In addition, previous studieshave demonstrated that disruption of the microtubules and ofthe microfilaments bring about alterations in the invasion andmetastatic behavior of neoplastic cells (17,18, 22) and that highand low metastatic cells differ in their pattern of cytoskeletonorganization (24, 32). In the present study, we have shown thatin B16 melanoma cells in which the organization of the intermediate filament network was altered by CH there was a reversibledecrease in the number of lung nodules. Recently, it was alsoshown that CH treatment markedly inhibits in vitro the invasionof M5076 tumor cells through the basement membrane of theamnion (31). However, while the decrease in vimentin synthesisobserved in CH-treated B16 melanoma cells is accompanied by

a decrease in the number of lung nodules, in B16 melanomacultivated in suspension, where we also found a decrease invimentin synthesis, the metastatic capability was increased (26).It appears therefore that while the state of organization of thecytoskeleton and the expression of the cytoskeletal proteingenes are linked, there is no simple correlation between theseand the expression of the metastatic capability. Nevertheless,this study further supports the notion that the cytoskeleton ismost probably involved in many processes fundamental to thecompletion of successful tumor dissemination (17, 18, 22). Thissuggestion is also supported by the control experiments in thepresent study where ara-C, that arrests DMA synthesis but does

not alter the cytoskeleton organization, also did not alter themetastatic capability of B16 melanoma, while treatment with CHthat disrupted the vimentin network also altered the metastaticpropensity of the cells. It is conceivable that the changes introduced in the cytoskeleton organization during suspension cultureand those induced by treatment with CH are very different andtherefore elicit different effects on the tumor dissemination capability of B16-F1 melanoma cells. It is also possible that since

CH has multiple effects on the cells, although the major cytoskeletal effect is specific to vimentin, the cell-colonizing proper

ties may be parallel but not due to the effect on vimentin. Sincemetastasis depends on the successful completion of several

linked sequential steps, it is difficult to evaluate the responsesobtained by isolated in vitro investigations especially due to thepossible host-tumor interactions that are important factors in

determining tumor dissemination.

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accumulation in human breast cancer cells (T-4D7) by human prolactin and 33. wittelsberger, S. C., Kleene, K., and Penman, S. Progressive loss of shape-growth hormone. Cancer Res. , 44: 1178- 1186, 1984. responsive metabolic controls in cells with increasingly transformed phenotype.

30. Takenaga, K. Enhanced metastatic potential of cloned low-metastatic Lewis Cell, 24: 859-866. 1981.

Fig. 1. Disruption of vimentin-containing filaments in cells treated with CH. CV-1 (A'), 3T3 (B'). and MDBK cells (C') which were treated with CH for 6 h or were left

untreated (A to C) were stained with anti-vimentin antibody, x 650.

Fig. 2. The effect of CH on intermediate filaments does not affect microfilaments and microtubules. BSC-1 cells which were treated for 6 h with CH (A ' to C ') or wereleft untreated to serve as control (A to C) were stained with anti-vimentin antiserum IA and A'), anti-tubulin antiserum (B and B'), or with rhodamine-phalloidin (C and C')

to visualize actin filaments, x 650.

Fig. 3. The effect of CH treatment on the vimentin network and cell shape in B16-F1 melanoma cells. B16-F1 melanoma control cells (A and A') or cells treated withCH for 4 h (B and B') or 8 h (C and C') or allowed to recover for 16 h after 8 h treatment with CH (D and D') were stained with anti-vimentin antibody (A to D), or thelive unfixed cells were photographed (A ' to 0 '). A to D, x 450; A ' to D ', x 105.

Fig. 4. The effect of CH treatment on vimentin synthesis in various cells. BSC-1, CV-1, MDBK, and B16-F1 melanoma cells and 3T3 and human foreskin fibroblasts(HFF) were treated for 6 h with CH, washed extensively, and pulse labeled in [^SJmethionine containing medium (Lanes 2). Alternatively, the cells were allowed torecover from the CH treatment for 8 h before pulse labeling with [MS]methionine (Lanes 3), or control untreated cells were pulse labeled for 1 h with ["SJmethionine(Lanes 7). Equal cpm from the Triton X-100-insoluble fraction were analyzed on SDS gels, a, actin; v, vimentin.

Fig. 5. Gradual decrease in vimentin synthesis following treatment with CH in B16 melanoma cells. B16-F1 melanoma cells treated with CH for 2 (Lane 2), 4 (Lane 3),6 (Lane 4), 8 (Lane 5), 11 (Lane 6), 16 (Lane 7), or after 16 h of treatment were incubated in fresh medium for 8 h (Lane 8). Following these treatments, the cells werewashed and pulse labeled with [^Slmethionine as described in Fig. 4, and the Triton X-100-insoluble cytoskeletal proteins were analyzed. Lane 7 and A. control untreatedcells; B, 6-h CH-treated cells; C, 6-h CH-treated cells followed by 16 h incubation in fresh medium. Equal trichloroacetic acid-precipitable cpm of the Triton X-100-insolublefractions were analyzed by SDS gels (Lanes 7 to S) or by 2-dimensional gel electrophoresis (A to C). a, actin; v, vimentin.

Fig. 6. Decrease in the mRNA concentration of cytoskeletal proteins in CH-treated cells. Poly(A)* cytoplasmic RNA from 6-h CH-treated B16-F1 cells (B) and untreatedcells (A) was translated in vitro in a reticulocyte lysate, and the proteins were analyzed by 2-dimensional gel electrophoresis. The Triton X-100-soluble fraction of ["S]methionine pulse-labeled control B16-F1 cells (C) or of 6-h CH-treated cells (D) was analyzed by 2-dimensional gel electrophoresis. Poly(A)* cytoplasmic RNA isolated

from control untreated cells (Lanes 7) or from cells treated with CH for 4 (Lanes 2), 8 (Lanes 3), 11 (Lanes 4), and 11 h followed by 8 h of recovery in fresh medium (Lane5) was analyzed on agarose gels, and RNA blots were hybridized to dCTMP-labeled complementary DNA clones containing vimentin (£),tubulin (F), or actin (G)sequences. The position of the cytoskeletal proteins on SDS gels and 2-dimensional gels was identified by immunoblotting with the respective antibodies, a, actin; v,vimentin; f,, i2, «and d tubulin; large arrowhead, unidentified protein, kb, kilobase.

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1985;45:2632-2641. Cancer Res   Avri Ben-Ze'ev and Avraham Raz  Vimentin and the Metastatic Capability of B16 Melanoma CellsRelationship between the Organization and Synthesis of

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