American Journal of Patholog, Vol. 140, No. 6, June 1992Copyright ©) American Association of Pathologists

Mutated C-Ha-ras Oncogene AltersCytokeratin Expression in the HumanBreast Epithelial Cell Line MOF-10A

Terry Maloney Paine,* Gabriella Fontanini,*Fulvio Basolo,*t Irma Geronimo,*James W. Elliott,* and Jose Russo*From the Michigan Cancer Foundation,* Detroit, Michigan;and The Italian Association for Cancer Research, Pisa, Ita4l

MCF-IOA, a spontaneously immortalized humanbreast epithelial cell line, has been transformed bytransfection with the mutated c-Ha-ras oncogene ob-tained from T24 carcinoma cells. The pattern of cy-tokeratin expression was studied in MCF-IOA cells incomparison with plasmid transfected or MCF-IOAneo cells, normal ras proto oncogene transfectedor MCF-IOAneoN cells, and transformed or MCF-IOAneoT cells. Cytokeratin expression was studied bywestern immunoblot of total cellular proteins sepa-rated by two-dimensional gel electrophoresis. Blotswith cytokeratin specific AEJ and AE3 antibodiesshowed identical molecular weight species of cytok-eratins in MCF-IOA, MCF-IOAneo, MCF-IOAneoN,and MCF-IOAneoT cells; however, in MCF-IOAneoTcells, the intensity of immunostaining and the num-ber of immunoreactive phosphorylated polypeptideskeratins 7, 8, 15, and 16 was decreased. It was con-cluded that c-Ha-ras oncogene-induced transforma-tion alters quantitatively the cytokeratin pattern ofhuman breast epithelial cells and that these changescould explain some of the morphologic alterationsobserved in c-Ha-ras transformed cells. (AmJPathol1992, 140:1483-1488)

MCF-1 OA is a spontaneously immortalized human breastepithelial cell line that has been maintained in culture formore than 4 years. 1'2 This cell line, which has retained itsnormal phenotype through the years in culture, upontransfection with mutated c-Ha-ras oncogene showsphenotypical characteristics of malignant transformationsuch as anchorage independent growth, forms coloniesin methocel without addition of hydrocortisone, choleraenterotoxin or epidermal growth factor (EGF), and ex-

hibits altered three-dimensional growth in collagen.Transformed cells also form tumors in nude mice.3'4 Dis-tinctive features induced by the mutated c-Ha-ras onco-gene were the lengthening and thickening of cell surfacemicrovilli, formation of blebs, and emission of filopodialprojections. It induced cytoplasmic changes consistingin formation of intracellular lumens, and increased thenumber of lysosomes, mitochondria, and glycogen con-tent, significantly decreasing the number of intermedi-ated filaments.35

To further characterize the alterations of the normalphenotype induced by transfection with the mutatedc-Ha-ras oncogene, we have studied in MCF-10A theexpression of cytokeratins, a family of intermediate fila-ment proteins that are cytoskeletal components of virtu-ally all epithelial cells. On the basis of two-dimensional gelelectrophoresis, Moll and colleagues have cataloged 19human epithelial cytokeratin polypeptides,5 which aresubdivided according to their charges and immunoreac-tivities as acidic (type 1) or basic (type 11).6 The cytoker-atins are differentially expressed during epithelial differ-entiation and are useful as markers for diagnosis of epi-thelium-derived neoplasm.7-9 Cytokeratins 5, 7, 8,14,15,17, 18, and 19 have been identified in the normal humanmammary gland,5 and reduction of the normal cytokera-tin profile to expression of cytokeratins 8, 18, 19, andsometimes 7, in mammary carcinomas and in breastcancer cell lines has been reported.10

Since it has been observed that the profile of cytok-eratin expression in malignant breast epithelial cells inculture is similar to that observed in vivo,10 and recentstudies have shown that several breast carcinoma celllines and one line of mammary epithelial cells trans-formed by transfection with v-Ha-ras and SV40 antigen

Supported by National Cancer Institute (CA 38921 to JR), NIH BiomedicalResearch Support grant (RR 05529) to the Michigan Cancer Foundation,NIH Cancer Center Core grant (CA 22453) to the Comprehensive CancerCenter of Metropolitan Detroit, and an institutional grant from the UnitedFoundation of Greater Detroit.

Accepted for publication January 13, 1992.Address reprint requests to Dr. Jose Russo, Department of Pathol-

ogy, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA19111.

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1484 Paine et alAJPJune 1992, Vol. 140, No. 6

(184A1N4-T-ras) exhibit cytokeratin downregulation,11we considered it important to study the expression of thismarker in the process of cell transformation. We usedtwo-dimensional gel Western blot analysis for determin-ing whether a single event, in this case the introduction ofa single oncogene in MCF-1 OA, which suffices to trans-form these cells, also elicits changes in the expression ofthe cytokeratin proteins.

Materials and Methods

Cells

The origin and development of the immortalized humanbreast epithelial cell line MCF-1i A, as well as its trans-fection with the mutated c-Ha-ras oncogene have beendescribed elsewhere.1- Cells transfected with plasmidcontaining the neomycin resistant gene, MCF-10A-neo,with the normal Ha-ras proto-oncogene (MCF-1 OA-neoN)or with the human T-24 mutated Ha-ras oncogene (MCF-1OA-neoT)3 were maintained in Dulbecco's minimal es-sential media (DMEM)/F-12 medium with 5% equine se-rum, 0.1 ,ug/ml cholera enterotoxin, 10 ,ug/ml insulin, 100U/ml penicillin, 100 ,ug/ml streptomycin, 2.5 ,ug/ml am-photericin B (Gibco, Long Island, NY), 0.5 ,ug/ml hydro-cortisone (Sigma Chemical Co., St. Louis, MO), and 0.02,ug/ml epidermal growth factor (EGF) (Collaborative Re-search, Palo Alto, CA).

Cell lines with known polypeptide composition5 usedas controls for immunostaining were MCF-7 breast car-cinoma cells, routinely maintained at the Michigan Can-cer Foundation; and A431, epidermoid carcinoma cells,obtained from the American Type Culture Collection(ATCC, Rockville, MD) and grown under ATCC recom-mended conditions.

by suspending the cells in lysis buffer; equal concentra-tions of lysate (100 ,ug), as determined by Coomassieblue dye-binding method, were separated on two-dimensional (2-D) gels as previously described,12 withisoelectric focusing on pH 3.8-7.5 gradients in the first-dimension tube gels and molecular size separation onsecond-dimension 10% polyacrylamide SDS slab gels towhich prestained molecular weight standards (SigmaChemical Co., St. Louis, MO) were applied. Proteins weretransferred electrophoretically onto a 0.05 ,um nitrocellu-lose membrane (Schlicher and Schuell, Keene, NH). Thetransfer was done overnight at a total of 510-525 Vhr(0.05-0.25 amps) in a 25 mmol/l Tris/190 mmol/I glycinebuffer with 20% methanol at 40C.13 The blots were pres-tained to reveal the total protein profile with a 1:1 dilutionof colloidal gold stain.14 The gold stain, which yields apink-gold color does not inhibit antibody binding, andfacilitates identification of immunoreactive polypeptidesin complex patterns. If proteins are heavily loaded, pre-dominant proteins such as actin exhibit a "halo effect"when gold-stained (i.e., the edges of the spot stain, butthe center of the spot may not stain). Small sections cutfrom the prestained blots were incubated overnight atroom temperature with AED and AE3 antibodies diluted1:500. The secondary antibody, peroxidase-conjugatedanti-mouse IgG (ICN Biomedicals, Inc., Lisle, IL) was di-luted 1:500 and applied for 4 hours.

For color detection of the immune reaction, horserad-ish peroxidase (HRP) color development reagent con-taining 4-chloro-1-napthol (BioRad Laboratories, Rich-mond, CA) which yields a purple color that contrasts withthe pink-gold stain of the proteins, was used. The immu-noblots were analyzed using a computer-based imageanalysis system,15 which segments polypeptide spotsfrom background, records their position, measures theirintegrated densities (amounts), and allows comparison oftwo gels by superimposing images.

Antibodies

The mouse monoclonal antibodies (MAbs) to cytokera-tins AE1 and AE3 (ICN Biomedicals, Inc., Lisle, IL)79were used. AE1 recognizes most of the acidic (Type I)cytokeratins, numbers 10, 14, 15, 16, and 19; AE3 rec-ognizes all of the basic (Type 11) keratins, numbers 1-8,

Two-dimensional Ge/ Western Blotting

MCF-10A cells between passages 105 and 135 andtransfected cells at passages 16-20 post-transfectionwere used in these experiments; MCF-7, a malignantbreast cancer cell line that expresses keratins 8, and 19;19 was used as a control. Total cellular protein from cellsin log-phase of growth (80% confluence) was extracted

Results

Two-dimensional gel Western blot profiles of cytokeratinpolypeptides of total cellular extracts from parental MCF-1CA cells, its Ha-ras transfectants, and MCF-7 cells, de-tected by monoclonal antibodies AE1 and AE3 specificfor acidic and basic cytokeratins, respectively, are shownin Figure 1. The polypeptide profile within Mr 60,000 -36,000 (pH 4.5-6.8) is shown. Immunoreactive polypep-tides include different isoelectric variants of the same cy-tokeratin, as well as forms of lower molecular weight,which may have undergone degradation or some otherpost-transcriptional modification. In series of isoelectricvariants, all but the most basic spot usually representphosphorylated modifications.5 The immunoreactive

ras-altered Cytokeratins in Breast Cells 1485AJPJune 1992, Vol. 140, No. 6

Acidic cytokeratins (AEl) Basic cytokeratins (AE3)

M OF-1QA-

MCF- 1 0neo

MCF-IO neoN

M OF-lO neoT

M4CF-7

Figure 1. Identification of cytokeratins in 2-D gel Western blotsfrom MCF-10A transfected cells, and MCF-7 cells; 100 pgof total cellular

Protein were seParated on 2-D gels and transferred to nitrocellulose. Polypeptide profiles u'ere visualized uwith gold stain, then appropriate

areas of the blot were cut out and immunoreacted with anticytokeratins. The polvpeptide profile within Mr 60,000-36,000/lpH 4.5-6.8 is

shown. LowpH is at the left of each blot; a, actin, Mr 43,000/pH 5.4 has a negatively stained appearance due to its high concentration, and

is shown for reference. The immunoreactive polypeptides appear more darkly stained, against a background of lighter, nonreactive poly-

ytokeratin is labelled according to the nomenclature ofMoll

keratins 15 and 16 in MCF-10A MCF-1OAneo, MCF-1OAneoN and MCF-1OAneoT, and in MCF-7, keratin 19 (left panel). Antibody AE3

detected keratins 7 and 8 in MCF-10A MCF-l1OAneo, MCF-I1OneoN, and MCF-1OAneoT, and in MCF- 7 keratin 8 (right panel).

polypeptides were identified by molecular weight andapproximate pH (Table 1) as cytokeratins 7, 8,15, and 16according to the nomenclature of Moll et al,5 and by com-parison to immunoreactive polypeptides of A431, knownto express cytokeratins 5, 6, 7, 8, 13, 14, 15, 16, 17, and18. Cytokeratins 7, 8, 15, and 16 were present in MCF-10A cells and in all the transfectants; whereas MCF-7

cells expressed cytokeratins 8 and 19, this latter beingabsent in MCF-1 OA and transfectants (Figure 1, Table 1).Cytokeratin 14 was detected in MCF-1 OA cells inconsis-tently, and at low levels, and not in the transfected cells(Table 1).

Even though MCF-10A and transfectant cells ex-pressed identical cytokeratin patterns, the number of im-

1486 Paine et alAJPJune 1992, Vol. 140, No. 6

Table 1. Identification of Cytokeratins Recognized ey AE1 andAE3

AE1, acidic (type 1) AE3, basic (type II)*

Numbert 10 14 15 16 19 4 7 8Mol wt (x103) 56.5 50.0 50.0 48.0 40.0 59.0 54.0 52.0pI 5.3 5.3 4.9 5.1 5.2 7.3 6.0 6.1MCF-1 OA - + + + - - + +MCF-1 OAneo - - + + - - + +MCF-1 OAneoN - - + + - - + +MCF-1 OAneoT - - + + - - + +MCF-7 - - - - + - - +A-431 - + + + - - + +

* Cytokeratins 1, 2, 3, 5, and 6 (pl's >7.5) are recognized by AE3, but exceed the pH gradient of the two-dimensional gel system.t Cytokeratins are identified by their molecular weight, pl (isoelectric point) and Moll's catalog number.5+, present; +, weakly reactive and inconsistently present; -, absent.

munoreactive polypeptides of cytokeratins 7, 8, 15, and16 varied among the different cell lines. The intensity andthe number of immunoreactive polypeptides did not varysignificantly between MCF-1 OA and MCF-1 OAneo or

neoN cells, but the intensity of immunoreactive polypep-tides of 15 and 16 and the number of immunoreactivepolypeptides of 7 and 8 diminished in MCF-10Aneo Tcells (Figure 1, Table 2).

Discussion

Our results show that introduction of c-Ha-ras oncogenein MCF-1 OA cells reduces expression of cytokeratins 7, 8,15, and 16, as determined by 2-D gel Western blot anal-ysis. Whereas the normal ras gene is unable to inducesignificant changes in the expression of this cytoskeletalcomponent, the mutated ras oncogene significantly re-

duces 1) the number of polypeptides of acidic cytokera-tins 15 and 16, and 2) the intensity of polypeptides ofbasic cytokeratins 7 and 8 (Table 2).

Cytokeratins, the major cytoskeletal components of allepithelial cells, are encoded by a family of more than 20genes that are differently expressed in various types ofepithelia as well as in specific epithelial cells within a

given tissue;5'16 under normal conditions, cells appear tocoordinately express keratins type and 11.7

The epithelium of mammary gland ducts contains cy-

tokeratins 5, 7, 8, 14, 17, 19 and as minor componentscytokeratins 15 and 18,17 as revealed by gel electropho-resis. Cytokeratins 4 and 6 are not found in isolated duc-tal epithelium.18 The cytokeratin pattern is largely con-

served after transformation of epithelial cells, and it hasbeen shown that primary mammary carcinomas and theirmetastases have virtually the same cytokeratins as nor-

mal breast epithelium.17 Whereas several studies havebeen attempted to use keratins 5, 13, and 14 as markersof specific epithelial cells in the mammary glands,"'2-the pattern is not maintained in the cells in culture. Thisobservation also applies to cytokeratin 19, which hasbeen shown to be positive selectively for ductal epithelialcells7'21'23 and has been shown to be one of the majorcytokeratins of mammary gland tissue; however, it is notexpressed in MCF-10A cells, a finding reported by 2-Dgel and Western blot analyses23 and by immunocyto-chemical analyses in human breast epithelial cells de-rived from organoids.10 Cytokeratin 14 has been re-

ported to be present in the epithelial cells of large mam-mary ducts and also in myoepithelial cells.10 Weconfirmed this distribution in breast tissue from which

Table 2. Quantitation ofAE1 and AE3 Immunoreactive Polypeptides

AE1 AE3

Intensity Immunoreactive polypeptides Intensity Immunoreactive polypeptides

MCF-10A 7.3 ± 1.2* 14.0 ± 0.Ot 18.4 ± 1.8 34.0 ± 8.5tMCF-1OAneo 8.4 ± 6.2 8.5 + 3.5 12.1 ± 0.6 33.5 ± 6.4MCF-1OAneoN 7.0 ± 2.2 9.5 ± 2.1 14.9 ± 1.7 32.0 ± 12.7MCF-1OAneoT 3.7 ± 3.Ot 3.5 ± 0.7"1 6.3 ± 2.7¶ 19.5 ± 3.5#

* Intensities of individual polypeptide spots were expressed as a percentage of the total integrated density of all polypeptide spots on theimmunoblot (gold-stained and HRP-reactive), summed, and expressed as the mean ± SD of two experiments.

t Mean number of immunoreactive polypeptides ± standard deviation. One-way analysis of variance, Tukey's multiple comparison pro-cedure was used to determine statistical significance of differences in a and b.

t Not significantly different from MCF-10A and MCF-10AneoN (P = 0.6364)."Significantly different from MCF-1 OA (P = 0.033).¶ Significantly different from MCF-10A and MCF-1OAneoN (P = 0.012).# Not significantly different from MCF-10A and MCF-1OAneoN (P = 0.3482).

ras-altered Cytokeratins in Breast Cells 1487AJPJune 1992, Vol. 140, No. 6

MCF-10 cells were derived,2 and cytokeratin 14 ispresent in parental (mortal) MCF-10 cells,23 though it isinconsistently expressed at a barely detectable level inMCF-1i A cells, and not at all in the transfectants.

In human breast carcinoma, expression of cytokeratin14 by the myoepithelial cells was only found in associa-tion with the expression of collagen VII, a component ofthe basement membrane. Collagen VIl has been dem-onstrated to be the major protein in the anchoring fibrilsprojecting from the lamina densa into the subjacent con-nective tissue.21 Therefore, the lack of an appreciableamount of cytokeratin 14 in MCF-1 OA cells and its trans-fectants could be related to the lack of collagen VII orbasement membrane material. In the present work wehave used antibodies AE1 and AE3, which have beenwidely used to study the distribution of cytokeratins withintissues. AE1 antibody reacts with a subfamily of cytoker-atins that are acidic (type 1), whereas the AE3 antibodyreacts with a basic subfamily (type 11).7 Among the acidictype, we have identified by Western blot in MCF-1 OA cellsand its transfectants cytokeratins 15 and 16, whereaskeratin 19 is absent in these cells, but present in MCF-7cells. Even though mutated c-Ha-ras transfected cells ex-pressed the same cytokeratins as MCF-1OAneo andneoN cells, the intensity of the spots and the number ofimmunoreactive polypeptides both were reduced by theoncogene. Immunocytochemical analysis also showed asignificant decrease in staining throughout the differentpassages tested (results not shown). The basic or type IIcytokeratins present in MCF-1i A cells and transfectantsare cytokeratins 7 and 8, the intensity of which were sig-nificantly reduced in the cells transfected with the mu-tated ras gene. Another change observed was the re-duction in number of immunoreactive polypeptides inMCF-10A from 34 to 19.5 in MCF-10neoT; this was adecrease in phosphorylated forms, and in forms of lowermolecular weight, which may have undergone degrada-tion. The decreased number of polypeptides immunore-active with the cytokeratin antibodies may reflect a down-regulation in the expression or changes in phosphorylat-ed pattern of these proteins.

The mutated ras gene is able to induce an alteration inthe phosphorylation pattern24 since it interferes with twometabolic pathways that are known to control intracellularprotein phosphorylations: it downregulates enzymatic ac-tivities of the adenylate cyclase (cAMP pathway)25 andactivates the phosphotidylinositol turnover.26 Cellularevents such as growth and differentiation are controlledby phosphorylation/dephosphorylation of structural pro-teins and enzymes. Many oncogenes encode proteinswith kinase activity which phosphorylate target proteins;ras p21 possesses GTP-binding24 and GTP-ase27 ac-tivity.

Our observation that ras transfection results in a de-crease in the amount of the detected cytokeratins is sim-ilar to results obtained with another class of intermediatefilament proteins, vimentin. Phosphorylation of vimentinby the v-mos encoded kinase of Moloney sarcoma virus-infected cells induced disassembly of vimentin filamentsand resulted in a reduced amount of vimentin, whichcleaved to form a product with a more basic isoelectricpoint and lower apparent molecular weight.28 Alterationin the expression of cytokeratins during transformationhas been reported extensively.52934 Additionally, ourprevious reported observation of reduction or disappear-ance of tonofilament bundles in ras-transformed MCF-1 0A35 is explained by the reduction in the number ofcytokeratin polypeptides observed in the immunoblots,which in turn could be attributed to alterations in phos-phorylation pattern.

Acknowledgments

The authors thank Dr. Irma H. Russo for critical review of themanuscript, Ms. Karen Stephens for excellent technical supportin preparing the 2-D gels, Mr. Paul Juneau for biostatistical anal-ysis, and Ms. Vivian Powell for manuscript preparation.

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