Reduced Trisialoganglioside Synthesis in Chemically but not ......1L. Srinivas,unpublisheddata. 8Numbers in parentheses, total cpm incorporated into gangliosides per 10* cells. JB6

[CANCER RESEARCH 44,1510-1514, April 1984]

Reduced Trisialoganglioside Synthesis in Chemically but not mos-

transformed Mouse Epidermal Cells

LéelaSrinivas and Nancy H. Colburn1

Cell Biology Section, Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701

ABSTRACT

A specific decrease in the net de novo synthesis ([1-14C]-

glucosamine incorporation) of cell surface trisialoganglioside (GT)occurs in preneoplastic mouse JB6 epidermal cells in responseto tumor-promoting phorbol esters, mezerein, or epidermal

growth factor, all of which promote neoplastic transformation inJB6 cells, but not in response to the bladder promoter sodiumcyclamate, a nonpromoter in JB6 cells. The ganglioside showingelevated synthesis after mezerein or epidermal growth factorexposure is monosialoganglioside 1, whereas disialoganglioside1b synthesis is elevated after phorbol ester exposure. Primarymouse epidermal cells and putatively initiated epidermal cell linesselected for their resistance to induction of terminal differentiationby high calcium are resistant to promotion of anchorage-independent transformation by 2-week exposure to 12-O-tetradeca-noylphorbol-13-acetate. In both cell types, little or no decreasein GT synthesis occurs in response to short-term 12-O-tetradec-anoylphorbol-13-acetate exposure, thus extending further ourprevious observation that this GT response is restricted to pro-

motable cells. A decreased synthesis of GT also occurs consistently in cell lines transformed by 12-O-tetradecanoylphorbol-13-acetate or W-methyl-W-nitro-nitrosoguanidine as compared with

their nontransformed counterparts but not in cell lines transformed by a cloned integrated murine sarcoma provirus containing the oncogenic sequence v-mos. Thus, reduced cell surface

GT synthesis may be important both in the induction and in themaintenance of the chemically transformed but not viral oncogene mos-transformed phenotype in mouse epidermal cells.

INTRODUCTION

Gangliosides are integral components of plasma membranewhich are involved in regulating cellular growth and differentiation(16). When exogenously incorporated into normal or virus transformed cells, certain gangliosides reduce or delay cell growth(2), decrease saturation density (19, 20), or modulate the bindingof growth factors to cell surface receptors (4). In neuronal cells,gangliosides induce axon formation (24). We have reportedpreviously that JB6 mouse epidermal cells when treated with thetumor promoter TPA2 showed (a) promotion of anchorage inde

pendence and tumorigenicity (6, 7) and (b) a pronounced decrease in the de novo synthesis of GT (presumably GTib) asmeasured by [1-14C]glucosamine incorporation (28). The reduced

GT synthesis occurred with an onset of 4 hr and a maximal effectat 24 hr of TPA exposure (28). Both events were antagonized

' To whom requests for reprints should be addressed.2The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;

HPTLC, high-performance, thin-layer chromatography; GM, Go, GT, and Go,mono-, di-, tri-, and quatrasiatogangliosides with a T or "2" or "a" or "b" subscript

designating alternative positions of a sialic acid group; EGF, epidermal growthfactor; P~, promotion insensitive.

Received September 9, 1983; accepted January 10, 1984.

by the antipromoter retinoic acid (8, 28). This decrease occurredconsistently in TPA-sensitive promotable clonal cell lines but notin P~ variants (29), suggesting that this GT synthesis decrease is

required for induction of transformation in JB6 cells. Furthersupport for this possibility was obtained from the observationthat, when GT was added to cells treated with TPA, induction ofanchorage-independent transformation was inhibited (29). Thisinhibitory activity was shown by GT but not by any other sialo-glycocongugates tested (29). In this paper, we report the resultsof our experiments to determine (a) whether the GT responsegeneralizes to other promoters of transformation, (b) whetherthe lack of GT response generalizes to other independentlyderived promotion-insensitive cells, and (c) whether reduced GT

synthesis extends to neoplastically transformed cells.

MATERIALS AND METHODS

Cells. Promotable JB6 mouse epidermal cells (7, 10) were grown asdescribed previously (28). The development and characterization of JB6transformed lines are described by Colbum ef al. (5, 7, 11). Primaryepidermal cells from 1- to 3-day-old BALB/c mice were cultured in low-

calcium (0.07 rriM CaCI2 containing) Medium 199 obtained from NIH, asdescribed by Yuspa ef al. (33). Putatively initiated epidermal cell lines,LC7 (Footnote 3; Ref. 17) and 308 (32) that were resistant to inductionof terminal differentiation by a shift from low- to high-calcium-containing

medium, were kindly supplied by Dr. Henry Hennings and Dr. StuartYuspa of the National Cancer Institute. Line 308 was derived after invivo exposure to 7,12-dimethylbenz(a)anthracene and line LC7 after invitro exposure to solvent alone. JB6 C125, a P" cell line (12), was used

as recipient for transfecting pml v-mos DNA kindly supplied by Dr. Donald

Blair of the National Cancer Institute. The pm1, a pBR 322 subclone ofXm1 (30), is a cloned Moloney sarcoma virus DNA with terminallyredundant sequences. Transfection was carried out using calcium phosphate DNA precipitates and carrier DNA from P" cells (9). The v-mos

DNA-tranfected cells showed anchorage-independent growth in 0.33%

agar. These colonies were individually plucked from agar, and 2 of themwere grown as clonal lines designated C125/pm1-2 and C125/pm1-6.

Chemicals. Mezerein and TPA were obtained from Chemical Carcinogenesis, Eden Prairie, MN. EGF was kindly supplied by Dr. BruceMagun of the University of Arizona. Sodium cyclamate was purchasedfrom Sigma Chemical Co., St. Louis, MO. Ganglioside standards werepurchased from Supelco, Inc., Bellefonte, PA, and Silica Gel G thin-layer

chromatography plates were from Brink man Instruments, Inc., Westbury,NY. [1-14C]Glucosamine (55 mCi/mmol) was purchased from Amersham/

Searle Corp., Arlington Heights, IL. All solvents used were of analyticalgrade from J. T. Baker Chemical Co., Phillipsburg, NJ. Goib was agenerous gift of Dr. Robert Yu of Yale University. HPTLC plates werepurchased from E. Merck for the identification of unknown ganglioside.The identification of G, was based on HPTLC separation using 2 solventsystems (26).

Analysis of Ganglioside Synthesis. Cells were treated with TPA orother promoters at indicated concentrations for 24 hr during logarithmicgrowth with [1-"C]glucosamine (5 /iCi/ml) labeling for the terminal 4 hr

3 H. Hennings, personal communication.

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Ganglioside Synthesis in Transformed Cells

Table 1Effect of nonphorbol promoters on [ "C]glucosamine incorporation into gangliosidesof JB6 cells

JB6 cells were treated with mezerein, EGF, or sodium cyclamate for 24 hr with a terminal 4-hr labelingwith [1-14C]glucosamine(5 //Ci/ml). A0.1% dimethyl sulfoxide solvent control was run concomitantly. Gangliosideswere extracted as described previously (28)and separated by thin-layer chromatography using Silica Gel G plates with chloroform:methanol:ammoniumhydroxide:water (60:35:0.5:7.5, v/v), and gangliosideswerevisualizedby iodine vapor. The spots were scraped, extracted in 1.0 ml CHCI3:CH.,OH:H2O(10:10:1),and radioactivity was determined by liquidscintillation counting. Individual values are expressed as a percentage of total ganglioside radioactivity (cpm) recovered from thin-layerchromatography plates and are the mean of duplicate samples which differed by less than 10% from the mean.

% of total gangliosidecpm recovered

MezereinOWGcni.)GT

Gotb

GDI.GM,

GM2G«Total

gangliosidesUntreated

control18.0

52.025.02.01.0

<1.0<1.0100.0(4328)"10ng/ml

(16.2mM)38.8

4.014.63.0

36.92.0

<1.0100.0(2552)100ng/ml

(162nM)19.0

2.519.05.6

48.93.8

<1.0100.0(1521)EGF0.5

ng/ml(0.082nu)52.9

7.022.03.69.05.6

<1.0100.0(2436)1.0

ng/ml(0.164nM)67.0

1.51.09.0

17.73.2

<1.0100.0(1176)Sodium

cyclamate0.1%(2.01

HIM)40.3

38.220.72.12.0

<1.0<1.0100.0(3836)0.5%

(5.0mM)21.8

41.028.0

3.82.82.0

<1.0100.0(3785)

* Numbers in parentheses, total cpm incorporated into gangliosides per 10°cells.

of treatment. Comparison of 4-, 6-, 12-, and 24-hr incorporation of

precursor showed a constant distribution into individual gangliosides inuntreated cells and a constant but altered distribution of cpm incorporated during a 4- to 24-hr terminal period after 24-hr TPA exposure (not

shown). The cells were harvested, the gangliosides were extracted withchloroform-methanol and separated by thin-layer chromatography on

Silica Gel G, and the specific radioactivity of the gangliosides wasdetermined as described earlier (28).

RESULTS

Effects of Nonphorbol Tumor Promoters on GangliosideSynthesis. Three nonphorbol agents which have tumor-promot

ing activity in mouse skin or other systems were examined fortheir activity in inducing ganglioside changes, particularly reducedsynthesis of GT in JB6 cells (Table 1). These agents includemezerein, a second-stage mouse skin tumor promoter (27) and

promoter of transformation in JB6 cells (12); EGF, a mouse skincocarcinogen (25) and promoter in JB6 cells (12); and sodiumcyclamate, a bladder tumor promoter (18) and nonpromoter inJB6 cells (14). All 3 compounds produced a 2- to 4-fold distributional increase in incorporation of [1-14C]glucosamine into the

Gx band, which by comparison with authentic GQib on HPTLCanalysis appears to contain GQib.4The Gx response to mezerein

and sodium cyclamate appears to be biphasic. All 3 agentsproduced at least a 2-fold increase in incorporation into GDia at

the higher concentrations tested. Only mezerein and EGF produced appreciable decreases in the synthesis of GT. In contrastto TPA (Table 2), mezerein and EGF produced striking increasesin GMI synthesis. This phenomenon was not observed in sodiumcyclamate-treated cells. TPA, mezerein, and EGF, at the concen

trations shown in Table 1 and Table 2, showed high activity forpromotion of transformation in JB6 cells (12), while sodiumcyclamate showed none (14). Thus, the ganglioside changewhich correlates best with transformation-promoting activity for

these 4 compounds is the GT synthesis decrease.Ganglioside Synthesis Response to TPA in Primary Epi

dermal Cultures and in "Initiated" Calcium-resistant Epider

mal Cells. We next sought to ascertain whether the gangliosideresponses to promoters observed in the relatively homogeneous

Table 2Effect of TPAexposureon ganglioside synthesis in mouse epidermalprimary cells

and JB6 promotable mouse epidermalcell linesThe separation, visualization, and quantitation of ganglioside synthesis are

exactly as described in Table 1. Values are the mean of duplicate samples whichdiffered by less than 10% from the mean.

% of total gangliosidecpm recovered

PrimarycultureGangliosidesG,

(GQ1I))GTGoibGo,.GM1G«,GMTotal

gangliosidesUntreated36.0

46.02.62.09.32.2

2.1100.0(4473)"TPA

treated[10 ng/ml(1.6x10-'

M)]38.0

37.311.81.05.33.6

3.2100.0(2431)JB-6

promotablecellsUntreated18.0

62.016.21.2

<1.0<1.0<1.0100.0(3200)TPA

treated[10 ng/ml

(1.6X10-*M)]38.4

16.038.83.8

<1.0<1.0<1.0100.0(1524)

1L. Srinivas, unpublisheddata.

8 Numbers in parentheses, total cpm incorporated into gangliosides per 10*

cells.

JB6 epidermal cell line could also be observed in heterogeneouspopulations of primary epidermal cell cultures. Primary epidermalcultures are, except for lack of initiator treatment, presumablycloser to the population of cells exposed to TPA in mouse skintumor promotion experiments. Previous studies have shown thatepidermal cells from primary cultures are not inducible to anchorage independence by a 2-week exposure to TPA (5). Table2 shows the ganglioside synthesis profile of primary epidermalcells with and without TPA treatment in comparison with that ofJB6 cells. GT ¡sthe major ganglioside being synthesized in both.The TPA treatment produced only a 19% distributional decreasein [1-14C]glucosamine incorporation into GT in primary cultures,

as compared to the greater than 75% decrease produced in JB6cells. The incorporation into Band Gx which contains GQlb wasincreased by TPA in JB6 cells but not in primary cultures. GDibsynthesis increased substantially in both primary and JB6 cellswith TPA treatment. The rest of the gangliosides did not showsignificant changes. The synthesis of total gangliosides was 50%less in TPA-treated cells than in untreated controls for both

primary cultures and JB6 cells.To investigate further the relationship of GTresponse to stages

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L. Srinivas and N. H. Colburn

of promoter-induced preneoplastic progression, we studied the

effects of TPA on putatively initiated mouse epidermal cells LC7(Footnote 3; Ref. 17) and 308 (32), selected for resistance tohigh-calcium-induced terminal differentiation (Table 3). These 2cell lines were not promotable to anchorage independence by 2-

week exposure to TPA. GT was the major ganglioside beingsynthesized in LC7 and 308 cells. These cells showed no distributional decrease in GT synthesis and an overall synthesis ofgangliosides that increased in response to TPA. In fact, theabsolute incorporation of [14C]glucosamine into GT was in

creased 2-fold in 308 and 9-fold in LC7 cells by TPA treatment.

The relative synthesis of both GDI»and G, was decreased inboth calcium-resistant lines in response to TPA.

Ganglioside Synthesis in Neoplastically Transformed Cells.The ganglioside synthesis profile of transformed cells was deter-

Tabte3Effect of TPA on anchorage-independent growth and ganglioside synthesis in

calcium-resistant epidermal cellsLC7 and 308 cells were grown in minimal Eagle's medium containing 0.07 now

Cad;., treated with TPA at 10 ng/ml for 24 hr, and terminally labeled for 4 hr with[1-"C]glucosamine. The gangliosides were isolated, separated, and quantitated

according to the method of Srinivas and Colburn (28). Values are means ofduplicates which differed by less than 10% from the mean. Induction of agar colonygrowth by TPA was assayed as described previously (7). The results were identicalin agar containing high (1.2 mu) or low (0.07 mm) CaCI2. Cell viability of LC7 and308 in agar medium was high. The agar colony yield of all cells was <10 " with or

without TPA treatment.

% of total ganglioside cpm recovered

308 LC7

Gangliosides Untreated TPA treated Untreated TPA treated

G.(Go»)GTGo»GDI.GM.CM*20.774.81.53.10.10.29.676.89.70.81.31.833.047.04.34.95.23.216.280.00.50.12.60.2

mined to investigate whether reduced GT synthesis was associated with the neoplastically transformed phenotype (Chart 1). Inthe JB6-derived TPA-induced transformants, namely, T36274,

R681, and RT101, GTaccounted for only 2 to 7% of the precursorincorporation into gangliosides, as contrasted with 62% intoparental JB6 cells. In JB8 and VB8, 2 transformants paired withthe nontransformant JB1 by virtue of similar origin via A/-methyl-A/-nitro-nitrosoguanidine exposure of primary cultures, there was

also a substantial though less extensive decrease in GT synthesis. Thus, from Chart 1, it can be concluded that, for this set ofcell lines, reduced GT synthesis is consistently associated withthe chemically transformed phenotype. Since overall synthesisdecreased on transformation, the absolute decreases were evenmore pronounced. Increased GDia synthesis also appears to beassociated with chemical transformation in mouse epidermal celllines.

Having obtained data implicating reduced G, synthesis in bothinduction and maintenance of chemical transformation in JB6cells, we sought to determine whether reduced GT synthesiswas associated with another mode of transformation, namely,that induced by transfection of a viral oncogene mos. JB6 C125, a promotion-insensitive JB6 clonal cell line (12), was trans-

fected with pml Moloney mos DMA (9, 30), and the transformedcells were cloned from soft agar. When these transformed celllines were analyzed, G, synthesis as a percentage of totalganglioside synthesis was found to be about 35% lower thanwas untransfected controls (Chart 2). The incorporation of [1-14C]glucosamine into total gangliosides was elevated in the mos

transformants in contrast to the chemical transformants shownin Chart 1, yielding a small absolute increase in incorporation intoGT. GDU was the ganglioside the synthesis of which was consistently and substantially elevated in mos transformants, as wasalso observed for chemically transformed cells.

Total gangliosides

100.0(6099)' 100.0(12452) 100.0(1279) 100.0(6955) DISCUSSION

* Numbers in parentheses, total cpm incorporated into gangliosides per 10e

cells.

The nonphorbol promoters of transformation in JB6 cells mez-erein and EGF, like phorbol diesters, produced a substantial

Chart 1. Ganglioside synthesis in chemically transformed mouse epidermal cells. JB6 cells were obtainedas a long-term line from BALB/c mouse primary epidermal cultures as described previously (10) and werestably nontumorigenic and anchorage dependent for100 passages (5, 12). JB6 cells became irreversiblytransformable by tumor promoters to anchorage independence and tumongenioty by the 35th passage (7).The 5 transformed cell lines used were anchorageindependent and tumorigenic. T"6274 cells were de

rived from JB6 cells after 3 cycles of cloning in softagar with TPA induction (7). R681 and RT101 wereclonally derived after selection of JB6 C141 cells forTPA resistance (11). JB8 and VB8 were mouse epidermal cell lines obtained from BALB/c mouse primarycultures after N-methyl-W-nitro-nitrosoguanidine treatment (10). Logarithmically growing cells were labeledwith [1-'4C]glucosamine for 4 hr and gangliosides ex

tracted as described previously (28) and separated bythin-layer chromatography, the ganglioside bands visualized by iodine, spots marked and scraped, and radioactivity determined using liquid scintillation counting.The radioactivity associated with each band was expressed as a percentage of total radioactivity associated with gangliosides recovered from the piate. Thevalues represent the mean of duplicate dishes; bars,S.D. £1,nontransformed cell lines; D, transformed celllines. The incorporation of [ 1-"C Iglucosamine into totalgangliosides of each cell line was as follows in cpm/10"cells: JB6,4742; JB1,3684; T36274.2456; R681,2638;

RT101,1987; VB8, 2339; JB8, 2283.

Q.U

70

60

50

(Oo=5, 40

O

o

*

JB6JB1

R661

RT101

VB8

JB8

tlf234567

Gx|1 2 3 456

GT

2345

GDib

67

Õ[il

íl

12 345

Gola

67 123 4567GMI

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60Ganglioside

CPMgèS2

ôi-'S2010

n1mÎuÕÕll1.

CI252.CI25 p MM23.CI25 pM16Ì

i»TIÄ1 2 3 1 2 3 1 2 3 1 2 3

GT Goib Gola GM1

Chart 2. Ganglioside synthesis in mos-transformed mouse JB6 cells. JB6 C125,a TPA-nonpromotable cell line, was transfected (9) with Moloney mas pm1 (30)DMA containing the mos oncogene, and the transformed cells (O) were cloned fromsoft agar and grown as monolayer. These cells were then labeled with [1-14C]

glucosamine for 4 hr and the gangliosides were isolated, separated, visualized, andquantitated as described earlier (28). The total incorporation of [1-MC]glucosaminefor each cell line for 1 x 106 cells is as follows: JB6CI 25, 3600 cpm; CI 25/pm1-2,7177 cpm; CI 25/pm1-6, 6390 cpm. O. nontransformed cell line. Bars, S.D.

decrease in GTsynthesis in JB6 cells. These effects of decreasednet incorporation of [1-14C]glucosamine into GTcould result from

a deficiency of glycosyl transferases which catalyze the synthesisof the complex gangliosides from GMsor from increased breakdown of GTby neuraminidases induced or activated by the tumorpromoter. TPA, in contrast to mezerein and EGF, producedelevated synthesis of GDib, not of GMi- This suggests that thesepromoters utilize 2 different biochemical pathways to achievethe same end point of reduced GT synthesis. This action may becharacteristic of complete and second-stage promoters. Its sig

nificance may be involved in bringing the cell closer to thetransformed phenotype by simplifying the ganglioside profile (2,3,16).

GT was found to be the major ganglioside synthesized inprimary epidermal cultures, calcium-resistant putatively initiated

epidermal cell lines, and JB6 epidermal cell lines. The profile ofganglioside synthesis in these epidermal cells is significantlydifferent from that in various fibroblasts which synthesize GM3asthe major ganglioside and GT as a minor ganglioside (21).

TPA-treated P~ primary mouse epidermal cells showed a small

GT decrease, as compared to that which occurs in promotion-sensitive JB6 cells. This may reflect the heterogeneity of primarycultures. One subpopulation may undergo a large decrease inGT synthesis, in which case the GT decrease would be aninsufficient event for promotion of transformation. Alternatively,primary cultures could be homogeneous for the GT response butnonpromotable (5), because they lack a sufficient magnitude ofGT response. The 19% decrease in GT synthesis produced byTPA in primary epidermal cells is similar to that shown by 2 ofthe P~ clonal variants of JB6 cells (29). The primary cultures also

showed differences from JB6 cells in GDib and GMi synthesis inboth TPA-treated and untreated cells. This presumably reflects

the existence of cell subpopulations not present in JB6 cells.

Ganglioside Synthesis in Transformed Cells

The putatively initiated calcium-resistant mouse epidermal cells

showed neither a decrease in GT synthesis nor promotion ofanchorage-independent transformation in response to short-term

exposure to TPA. This observation suggests that the resistanceof these cells to promotion of transformation by 2-week exposureto TPA may be attributable to their lack of GT response. Thecalcium-resistant phenotype, which also characterizes JB6 P~cells, may represent an early stage, and the "G-r-responsive"

phenotype, which characterizes promotion-sensitive cells, may

represent a later stage in preneoplastic progression.The ganglioside synthesis profiles of A/-methyl-A/-nitro-nitro-

soguanidine- or TPA-transformed epidermal cell lines when com

pared with nontumorigenic JB6 or JB1 cells showed consistentlyreduced GT synthesis. The occurrence of low GT synthesis intransformed cells is compatible with the possibility that reducedGT synthesis is a required biochemical event for maintaining thetumor phenotype. Reduced net GT synthesis could occur as aresult of a specific hydrolysis by a neuraminidase. It has beenreported that viral transformation of cells can produce increasedneuraminidase activity in chick embryo fibroblasts (23). Thisincreased activity might lead to the simplified ganglioside profilefound in various transformed cells (2,3,16). We have preliminaryevidence to show the presence of a neuraminidase with somedegree of specificity for GT in JB6 cells. Such an enzyme activitycould be due to the existence of a specific protein activator, asreported for GMi and GM2degradation (22). Whether a neuraminidase is activated or induced due to TPA action or is active intransformed cells needs to be established.

In order to determine whether reduced GT synthesis generalizes to viral oncogene-transformed JB6 cells, 2 clonal lines ofpm1 Moloney mos DNA-transfected, anchorage-independenttransformants of a JB6 P~ cell line were studied. These differed

from TPA-transformed JB6 cells in showing a smaller magnitudeof distributional reduction in GT synthesis relative to nontransfor-

mants. These were similar to chemically transformed cells inshowing a substantial increase in GDi»synthesis. The totalganglioside synthesis was increased by about 2-fold after mos

transformation. Thus, GT synthesis underwent an absolute increase after mos transformation. These results suggest that viralmos DNA transfection induces transformation by a differentmechanism than does TPA, as reflected in the ganglioside synthesis by these transformants and/or that the basis for maintenance of neoplastic transformation is different in the 2 cases.Current studies in our laboratory are concerned with characterization and purification of genes that specify sensitivity to promotion of neoplastic transformation by tumor promoters (9).Initial evidence suggests that these genes are different from theoncogenes, including mos, that have been described to date (notshown).

That tumor promoters produce some of the same biochemicalchanges that are constitutively expressed in tumor cells hasbeen observed frequently (31). Among these changes, at least 2have been observed in JB6 cells as well as other systems,namely, reduced collagen synthesis (13, 14, 15) and reducedfibronectin synthesis and release (1, 15). Our current observations regarding reduced GT synthesis in chemical transformantsalso follow this pattern. However, the reduced GT responsediffers from the collagen and fibronectin synthesis responses inJB6 cells in that the GT response is specific for promotion-

sensitive cells.

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L. Srinivas and N. H. Colburn

ACKNOWLEDGMENTS

We thank Edmund Wendel and Roy Sims for excellent technical assistance; Dr.Fredenco Bertolero. National Cancer Institute, for primary epidermal cultures; Dr.Henry Hennings and Dr. Stuart Yuspa of the National Cancer Institute for calcium-

resistant cells; Dr. Donald Blair, National Cancer Institute, for the pM1 DNA;Catherine Talmadge for carrying out transfection of pm1 Moloney mos DNA; Dr.Howard Holden, Dr. Donald Blair, and Dr. Henry Hennings of the National CancerInstitute for their critical review of the manuscript; and Beverly Bales for typing.

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1514 CANCER RESEARCH VOL. 44

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1984;44:1510-1514. Cancer Res   Leela Srinivas and Nancy H. Colburn 

-transformed Mouse Epidermal CellsmosReduced Trisialoganglioside Synthesis in Chemically but not

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