Proc. Natl. Acad. Sci. USA Vol. 87, pp. 7360-7364, October 1990 Cell Biology

Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells (subtractive hybridization/polymerase chain reaction/carcinogenesis/extraceflular matrix/tumor suppression)

PAUL YASWEN*t, AMY SMOLL*, DONNA M. AND MARTHA R. STAMPFER*

PEEHLO, DOUGLAS K. TRASK§, RUTH SAGER§,

*Cell and Molecular Biology Division, Lawrence Berkeley Laboratory, Building 934, 1 Cyclotron Road, Berkeley, CA 94720; tDivision of Urology, Department of Surgery, Stanford University Medical Center, Stanford, CA 94305; and §Division of Cancer Genetics, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115

Communicated by Melvin Calvin, June 18, 1990 (receivedfor review March 29, 1990)

described (4). The established cell lines 184A1 and 184B5 were derived from reduction mammoplasty specimen donor 184 after in vitro exposure of primary cultures to benzo[a]pyrene as described (1). 184B5 cells were subsequently infected with Kirsten sarcoma virus carrying a baboon pseudotype and injected into nude mice. Cell line B5KTu was established from a tumor resected from one such nude mouse (2). A1N4-TH was derived by sequential infection of A1N4, a variant of 184A1, with viruses carrying the simian virus 40 (SV40) T and the Ha-ras oncogenes (5). All of the cells derived from specimen 184, except A1N4-TH, were grown in MCDB 170 medium supplemented as described (4). A1N4TH was grown in a serum-containing medium (5). Prostatic epithelial cell cultures were established from radical prostatectomy specimens and grown as described (6). Breast tumor cell lines were provided by Charles Dollbaum (Peralta Cancer Research Institute, Oakland, CA) and grown in Dulbecco's modified Eagle's medium supplemented with insulin and lo0 fetal bovine serum. Hs578T was grown in MCDB 170 with 0.5% fetal bovine serum. Where indicated, cells were grown for one passage in the presence of human recombinant transforming growth factor type 131 (TGF31) (3 ng/ml) (Genentech). For extracellular matrix studies, 0.5 ml of liquid Matrigel (Collaborative Research), a commercially prepared extract from the transplantable Englebreth-HolmSwarm (EHS) mouse sarcoma, was spread onto a 100-mm dish and allowed to polymerize at 37TC before plating cells on top of the gel. RNA Isolation and cDNA Synthesis. Cell or tissue samples were harvested in buffered guanidine thiocyanate solution and RNA was purified by CsCl ultracentrifugation (7). Poly(A)+ RNA was purified, when necessary, by two rounds of oligo(dT)-cellulose chromatography (8). cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase (BRL) according to the manufacturer's instructions. Subtractive Hybridization. cDNA prepared from 20 lzg of 184 (passage 10-13 of subconfluent cells) poly(A)+ RNA was enriched for sequences preferentially expressed in normal cells by two rounds of hybridization with a 5-fold mass excess of poly(A)+ RNA from B5KTu to a corrected Rot of 6000, followed by selection of single-stranded cDNA by hydroxylapatite chromatography (9). The resulting single-stranded cDNAs, enriched :'50-fold, were labeled with [32P]dCTP to high specific activities by the random-primer method (10) and used to screen =60,000 independent plaques of a 184 cDNA

A human cDNA library obtained from culABSTRACT tured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[alpyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to almodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type 13 increased its relative abundance. The protein encoded by NB-1 may have Ca2' binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined.

In an effort to comprehend better the processes whereby normal human mammary epithelial cells (HMECs) can be transformed, we have initiated a search for specific genes whose expression could be relevant to this process. In this study, we chose to select and identify gene transcripts that were down-regulated during HMEC transformation, the objective being to identify genes whose activity might be incompatible with or suppress tumorigenicity. We compared a normal diploid breast cell strain, 184, derived from a reduction mammoplasty, with a tumorigenic cell line, B5KTu, derived from 184 cells after immortal transformation following exposure to the chemical carcinogen benzo[a]pyrene (1) and subsequent malignant transformation following infection with Kirsten sarcoma virus (2). This system was chosen because (i) the epithelial cells derived from normal tissue were stably diploid with a finite life-span (3), (ii) the tumorigenically transformed derivative grew in the same serum-free medium at approximately the same rate as the normal cell strain with minimal genetic drift (2, 3), and (iii) abundant quantities of both normal and transformed HMECs could be obtained from a single individual, free of other cell types.

MATERIALS AND METHODS Cell Culture. Reduction mammoplasty tissue was processed to yield epithelial cell clumps (termed organoids) as

Abbreviations: HMEC, human mammary epithelial cell; EHS, extracellular matrix material from the Englebreth-Holm-Swarm transplantable mouse tumor; TGF, transforming growth factor; PCR, polymerase chain reaction; SV40, simian virus 40; GAPDH, glyc-

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

eraldehyde-3-phosphate dehydrogenase. tTo whom reprint requests should be addressed.

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library prepared in Agt1O (8, 11). One hundred plaques displaying the highest intensity of hybridization were replated at low density and screened with freshly labeled subtracted probe or probe made from B5KTu cDNA. Plaques that showed positive hybridization with the subtracted probe, but not the heterologous probe, were used to prepare phage DNA by the plate lysis procedure (8). The phage inserts were cut out with EcoRI and isolated by low melt agarose gel electrophoresis. Random-primed inserts were then used as probes in Northern analyses. Representative clones with distinctive expression patterns were subcloned into pGEM-3 and sequenced by the dideoxynucleotide chain-termination method (12). Sequence comparisons were performed by using the GenBank nucleic acid data base (Release 61.0). Northern Analysis. Ten micrograms of total RNA was denatured, fractionated by electrophoresis in 1.3% agarose/ 2.2 M formaldehyde slab gels, and capillary blotted onto Zeta-Probe nylon filters (Bio-Rad). Filters were baked for 2 hr at 80'C, prewashed in 0.1x SSC (ix SSC = 0.15 M NaCl/0.015 M sodium citrate)/0.5% SDS for 1 hr at 650C, and prehybridized in 50% formamide/5 x Denhardt's solution (ix Denhardt's solution = 0.02% bovine serum albumin/ 0.02% Ficoll/0.02% polyvinylpyrrolidone)/20 mM sodium phosphate, pH 6.5/denatured sonicated salmon sperm DNA (200 ttg/ml)/0.2% SDS. Hybridization with 32P-labeled probes (10) was for 16-20 hr at 420C. Filters were washed in 2x SSC/0.1% SDS at room temperature, followed by 0.1x SSC/0.1% SDS at 65-C and autoradiography. Polymerase Chain Reaction (PCR) Amplification of mRNA. Five micrograms of total RNA from each specimen was reverse transcribed into cDNA using oligo(dT) primers. Excess primers were removed by ultrafiltration through Centricon-100 microconcentrators (Amicon). cDNA was suspended in 0.5 ml of TE (10 mM Tris HCl, pH 7.5/1 mM EDTA) and 20 ,ul was used for each amplification reaction. Amplification was carried out in 100 ,ul of PCR cocktail [67 mM Tris-HCl, pH 8.8/16 mM (NH4)2SO4/3 mM MgCl2/200 ,uM each dNTP] with 50 pmol of each primer and 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Perkin-Elmer/ Cetus) overlaid with 30 ,u of mineral oil. Thirty cycles of amplification were routinely carried out in a DNA thermal cycler (Perkin-Elmer/Cetus) using a step program (94°C, 40 sec; 50°C, 2 min; 72°C, 3 min), followed by a 15-min final extension at 72°C. The oligonucleotide primers used to specifically amplify NB-1 cDNA were NB1B (5'-CCCTGCTCATCCAAGCCTCC-3') and NB1F (5'-CCGCGTCTCTTCTCTGCACT-3'). As an internal gauge of cDNA synthesis, the primers GAPDH5 (5'-TCTCCAGAACATCATCCCTG3') and GAPDH3 (5'-CTCCTTGGAGGCCATGTGGG-3') were used to detect glyceraldehyde-3-phosphate dehydrogenase transcripts in cDNA samples. 5'-End Amplification of NB-1 cDNA. To clone the 5' ends of NB-1 transcripts, a modification of the "anchored" PCR technique (13) was used. One microgram of 184 poly(A)+ RNA was denatured with 2 mM methylmercuric hydroxide (Serva) in 5.5 ,ul at room temperature for 10 min. The methylmercuric hydroxide was then neutralized with 1 ,lI of 500 mM 2-mercaptoethanol for 5 min. The treated RNA was used immediately for cDNA synthesis with the NB-1 specific 3' primer NB-1D (5'-GGl-l-GCCTGCAGTGGGAGC-3'). The cDNA was purified by ultrafiltration through a Centricon-100 and resuspended in 30 ul of lx tailing buffer (BRL) and 200 ,uM dATP. Fourteen units of terminal deoxynucleotidyltransferase (BRL) was added and the reaction was allowed to proceed for 2 min at 37°C. After heating for 15 min at 65°C, the reaction mixture was diluted with 470 ,ul of TE, and 20-ul aliquots were used for PCR amplification as described above, except that the annealing temperature was raised to 55TC, and the following primers were used: NB-1C, 5'-CGGATCCGTGTCATTCAGATGAGC-3' (50 pmol);

Proc. Natl. Acad. Sci. USA 87 (1990)

7361

(dT)l7 adaptor, 5'-GACTCGAGTCGACATC-

FF1 Fl1-3' (20 pmol); adaptor, 5' GACGAlH 1-1-1-1 TCGAGTCGACATCG-3' (50 pmol).

RESULTS Differential NB-i mRNA Expression in Normal, Immortalized, and Tumorigenic Cells. cDNA clones identified by subtractive hybridization between 184 and BSKTu could be grouped into three classes on the basis of Northern analysis. Partial sequence analysis of representative cDNA clones showed that approximately half corresponded to mRNA encoding fibronectin, while a second group corresponded to keratin 5 (14). One cDNA fragment showed no similarity to any previously reported sequences. This 350-base-pair (bp) cDNA hybridized specifically to a 1.4-kilobase (kb) mRNA expressed in the 184 cells (Fig. 1). Levels of the mRNA, designated NB-i, were '40-fold lower in the immortalized 184B5 line and were further reduced to barely detectable levels in tumorigenic BSKTu (Fig. 1). In contrast with the stepwise decrease observed in the 184B5 series, NB-i RNA was undetectable in immortalized 184A1 cells and in the tumorigenic A1N4-TH cells. Although detectable by Northern analysis in several independently derived normal HMEC strains, NB-i mRNA was undetectable in all the cultured human breast tumor cell lines examined, both estrogen receptor positive and negative (Fig. 2). The only other RNA in which NB-i expression was detected by Northern analysis was that of epithelial cells cultured from human prostate (Fig. 3). Highest levels ofNB-i transcripts were observed in epithelial cells cultured from the central zone of normal prostate, while lower levels were apparent in those cells cultured from the peripheral zone. The cells cultured from primary prostatic tumors represented mixtures of normal and abnormal epithelial cells. The relative contribution of each of the cell types to NB-i expression was undetermined. The finding that NB-i expression was unaltered in normal prostate cells infected, but not immortalized, by SV40 suggested that viral infection alone did not influence the expression of this mRNA. NB-i mRNA was undetectable in the immortal prostate cancer line PC3. NB-1 mRNA Expression in Vivo. In an effort to correlate the expression of NB-i mRNA in cultured cells with that of whole tissue, total RNA was isolated directly from both normal and malignant human breast tissue, as well as from the purified epithelial organoids from which strain 184 was

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FIG. 1. Expression of NB-1 mRNA in normal and transformed HMECs. A 350-bp cDNA clone corresponding to the putative 3' untranslated end of the NB-1 mRNA was hybridized with a Northern blot containing 10 ,ug of total RNA from the indicated HMEC cultures. EtBr shows total RNA in the original gel stained with ethidium bromide.

Cell Biology: Yaswen et al.

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Proc. Natl. Acad. Sci. USA 87 (1990)

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derived. NB-1 transcripts were not detectable in any of these samples by Northern analysis, although they were detectable in primary HMEC cultures (data not shown). To detect small amounts of NB-1 mRNA that could not be identified by Northern analysis, PCR amplification of total cellular cDNA was performed with NB-1-specific primers. The predicted 531-bp cDNA fragment was clearly demonstrable by ethidium bromide staining of amplified preparations from normal organoids as well as cultured normal cells (Fig. 4A). Southern blot analysis with an internal NB-1 probe confirmed that the amplified bands corresponded to authentic NB-1 transcripts and showed that a weaker band of the same size was present in the lane corresponding to primary breast tumor tissue (Fig. 4B). In comparison, no NB-1 PCR product was detectable in a cultured breast tumor cell line (Fig. 4B). PCR amplification of the same cDNA pools, using primers for the glycolytic enzyme GAPDH, showed that this mRNA species was amplified with equal efficiency in normal and tumor samples (Fig. 4C). A subsequent experiment (Fig. 4D) showed that NB-1 transcripts could be detected in unprocessed normal breast tissue, that NB-1 mRNA content was variable in four primary breast carcinomas, and that NB-1 mRNA was undetectable in four breast tumor cell lines. Although NB-1 transcripts were not detected by Northern analysis in other normal epithelial cell strains (epidermis, sweat gland, bronchus), breast fibroblasts, or tumor cell lines (cervix, bladder, lung, duodenum, pancreas, colon) (Fig. 2; data not shown), Z7

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FIG. 3. NB-1 expression in normal and tumor-derived prostate cells in vitro. Poly(A)+ RNA was purified from two different strains of epithelial cells grown from cancer-containing prostate tissue (PE1, PE2), a SV40-infected epithelial cell strain (SV-N), epithelial cell strains from the peripheral zone (NPZ) and the central zone (NCZ) of normal prostate and from tissue with benign prostatic hyperplasia (BH), a fibroblast cell strain from normal prostate, and a prostatic cell line (PC3). Five micrograms of each RNA was probed sequentially with NB-1 (A) and c-myc (B).

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C FIG. 2. NB-1 expression in normal and tumor human epithelial cells in vitro. Total cellular RNA from normal sweat gland epithelial cell strain HSG4; normal keratinocyte strain KZ-1; normal HMEC strains 161, 48R, and 184; breast tumor cell lines Hs578T, MCF7, ZR75-1, BT2O, BT474, BT468, BT549, MDA435, and MDA468; and cervical carcinoma cell line HeLa was probed with labeled NB-1 cDNA. Ten micrograms of RNA was loaded in each case with the exception of HSG4, where 6 ,ug was used.

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FIG. 4. PCR amplification of NB-1 transcripts. (A-C) Five micrograms of total RNA prepared from a postselection normal HMEC strain (184 pi1O-4), epithelial organoids purified from reduction mammoplasty tissue (184 organoid), a human breast tumor cell line (ZR-75-1), and an infiltrating breast ductal Carcinoma tissue specimen (T-2843) were reverse transcribed into cDNA using oligo(dT) primer. (A) NB-1 cDNAs in each of the preparations were amplified by using PCR and specific primers corresponding to the 3' end of NB-1. The DNA products were then separated by agarose gel electrophoresis and stained with ethidium bromide. (B) A Southern blot of the same gel prepared by standard methodology (8) was probed with an m200bp EcoRI/Pvu II NB-1 cDNA fragment. (C) GAPDH cDNAs in the same cDNA preparations were amplified by using GAPDH-specific primers as an internal control of PCR amplification. In additional NB-1 amplification assays, sample RNAs included those of unprocessed reduction mammoplasty tissue (Normal tissue), three additional -breast carcinomas (T3334, T2854, T2840), and three additional breast tumor cell lines (MCF7, BT20, Hs578T) (D), and normal breast, colon, skin, and cervix tissue (E).

PCR analysis revealed the presence of these transcripts in normal cervix and epidermal tissue (Fig. 4E). Identification of the Putative NB-1 Gene Product. Numerous termination codons in all six possible reading frames, and a potential polyadenylylation signal (AATAAA) followed by an 8-base poly(A) run at one end suggested that the initial NB-1 fragment corresponded to the 3' noncoding region of the mRNA. Using specific oligonucleotide primers based on the sequence of this fragment, we were able to clone an additional 950 bp of 5' sequence by the anchored PCR technique (13). The identity of the clones obtained was confirmed by hybridization to an independent NB-1 oligonucleotide and by Northern analysis. Sequence analysis of several of the longer cDNAs revealed a 447-bp open reading frame with extensive sequence similarity at both the nucleic acid and amino acid levels to calmodulin. At the nucleic acid level, the NB-1 open reading frame displayed 70%, 71%, and 80o sequence identity with the three known human calmodulin mRNAs (15-17) (Fig. SA). This sequence conservation did not extend into the noncoding regions (data not shown). The similarity between the translated amino acid sequence of NB-1 and human calmodulin was 85% over the length of the entire protein (Fig. SB). Modulation of NB-1 Expression. To better understand the observed differences in NB-1 expression in vivo and in vitro, we attempted to identify both soluble and insoluble factors that modulated the expression of this gene. Additions of fetal bovine serum, cycloheximide, or 12-0-tetradecanoyl-13acetylphorbol were without effect on short-term expression of NB-1 mRNA in 184 cells (data not shown). Human TGFj3 up-regulated the expression of NB-1 in 184, 184B5, and B5KTu cells, although no induction of NB-1 mRNA was detected in 184A1 or A1N4-TH (Fig. 6).

Cell

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Biology:

Yaswen et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

GGAGACAGCCCGCCGGCCGCCCGGATCTCCACCTGCCACCCCAGAGCTGdGACAGAGCCGGGCTGCGGCACTGGGAGGGAGACCCCACAGTGGCCTCTTCTGCCACCCACGCCCCCACCCCTGGC

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ATGGCCGACCAGCTGACTGAGGAGCAGGTCACAGAATTCAAGGAGGCCTTCTCCCTG;TTTGACAAGGATGGGGACGGCTGCATCACCACCCGCGAGCTGGGCACGGTCATGCGGTCCCTG -----T--T-------- C--A--A---A-TG-T-----------A ---------- A-----T--A-----C--T ---AC- A--AAAG--A--T--A--T------A----A-------T-----A--------A------ A-TG---------A--A--T--T--AT-A-------A-----T--T---ACT--A--A--AAAG--A--T--G--T--A---A-A--T--T

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FIG. 5. Nucleotide sequence and predicted amino acid sequence of NB-1 cDNA and comparison with human calmodulin. (A) Nucleotide sequence of NB-1 cDNA and alignment with the published sequences of a human liver calmodulin cDNA (CAM-L) (16) and two human teratoma cell line calmodulin cDNAs (CAM, CAM-A) (15, 17). Hyphens indicate nucleotide identity with NB-1 cDNA. (B) The deduced NB-1 amino acid sequence shown in one-letter code and alignment with that of human calmodulin. :, Identity; ., conservative amino acid change. Amino acids thought to be directly involved in Ca2l binding are underlined.

Growth on EHS has previously been shown to support differentiation of a variety of cell types (18, 19). This material contains laminin, entactin, collagen type IV, heparan sulfate proteoglycan, as well as other less well defined components. Normal HMECs plated on EHS at high cell densities formed cellular networks that slowly condensed to form tubular and globular structures with striking resemblance to endbuds in intact mammary gland tissue (P.Y. and M.R.S., unpublished data). When grown on EHS for 96 hr, these cells showed decreased levels of NB-1 mRNA transcripts (Fig. 6A). The magnitude of the decrease appeared related to the initial density at which the cells were seeded. Modulation of NB-1 mRNA by EHS appeared intact in the 184B5 and B5KTu cell lines, although they displayed a much more limited range of structure formation (Fig. 6B). cm-

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FIG. 6. Modulation of mRNA expression in normal HMECs (A) and transformed cell lines (B) by EHS and TGF,8. (A) Normal HMEC strain 184 was plated at 5.0 x 106 (Hi EHS) and 1.0 x 106 (Lo EHS) cells per 100-mm dish on EHS. Control and TGFB-treated cultures were plated at 5.0 x 105 cells per dish directly onto culture dishes. (B) Cell lines derived from the 184 cell strain were plated at 5.0 x 106 cells per 100-mm dish on EHS. Control and TGFf-treated cells were plated at 3-5 x 105 cells per dish. Cells were harvested for RNA at 96 hr (EHS) or at subconfluence (Control and TGFB).

DISCUSSION Calcium has been shown to be a key regulator of cellular differentiation in epithelial cells of the breast and skin (20, 21). Calmodulin, through its interaction with a large number of enzymes, is a primary mediator of cellular responses to Ca2' fluxes (22). Antagonists of calmodulin action have been shown to inhibit growth (23), while increased calmodulin levels have been correlated with decreased cell cycle transit time in some cell lines (24). Considering that the three calmodulin genes in humans diverge as much as is theoretically possible, while still encoding identical proteins (17), it is clear that the entire protein is stringently conserved to preserve proper function. The sequence of the NB-1 transcript is identical to that of a previously reported intronless calmodulin-like gene originally described as an unexpressed pseudogene (25). Although genomic sequences specifying calmodulin-like coding sequences that differ by more than three amino acids from the authentic protein have also been described in other vertebrates (26, 27), the NB-1 gene is the only one to our knowledge that has been shown to be expressed at the mRNA level. The extent of sequence similarity of the translated NB-1 open reading frame to human calmodulin is 85%. Both proteins are theoretically the same size and share in common most of the amino acids constituting four calcium binding domains. The probability that NB-1 encodes a Ca2+ binding protein that differs subtly from calmodulin provides an additional level of complexity for Ca2+ regulation of cellular processes. The NB-1 product may compete with calmodulin for this cellular mediator and may bind with different affinity to cellular substrates. Unlike calmodulin, NB-1 expression may be confined to certain subpopulations of epithelial cells. Although NB-1 mRNA is easily detectable in total RNA from cultured HMECs, it is present at relatively reduced levels in normal reduction mammoplasty tissue and in the purified organoids from which the cultured HMECs are derived. It is unlikely that the disparity of NB-1 expression in primary tissue versus cultured cells is due mainly to differences in proliferative

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Cell Biology: Yaswen et al.

state because expression of the mRNA is not significantly decreased in senescing cells where proliferation is minimal, nor is it affected by known inducers of growth-regulated mRNAs. Furthermore, exposure of normal HMECs to TGFB is accompanied by increased synthesis of NB-1 mRNA, while cell division ceases. NB-1 mRNA levels are highest in the normal HMEC population, which displays active long-term growth in MCDB 170. These cells have undergone a process of "self-selection," during which the majority of the cells cease proliferation (4). On the basis of intermediate filament protein expression, postselection HMECs may have attributes of multipotent precursor cells. They express keratins 5 and 14, which are characteristic of basal cells, but with time in culture show increased expression of keratins 8 and 18, which are characteristic of luminal cells (28). They also express vimentin, an intermediate filament component normally associated with mesenchymal cells and embryonic epithelial cells, which may indicate that they are related to a subset of basal epithelial cells in vivo reported to contain vimentin in addition to keratins (29, 30). NB-1 expression in HMECs can be manipulated in vitro. The work of others has shown that EHS supports the polarization and differentiated function of mammary epithelial cells from pregnant rodents (18, 31). 184 cells plated on EHS coalesce into tubular and globular structures and show decreased levels of NB-1 transcripts. This down-regulation of NB-1 expression in 184 or 184B5 cells shifted from plastic substrates to EHS occurs in a short time frame without observable cell selection. Whether the change is attributable to changes in cell shape and/or specific ligand-receptor interactions remains to be determined. Infiltrating breast ductal carcinomas most often exhibit a mature luminal phenotype in vivo and in vitro (28). The phenotype displayed is ordinarily associated with less proliferative normal cells in culture (28). Such an association in tumor cells may imply that the coordinated regulation of growth and differentiation has been disrupted, and/or that a particular differentiated state is more compatible with malignancy. Changes in signal transduction of growth or differentiation factors and their associated intracellular second messengers are often implicated in neoplastic transformation. The strong homology between NB-1 and calmodulin, combined with the regulated pattern of expression exhibited by the NB-1 gene, suggests that the NB-1 gene product is a signal transducer that may play a differentiation-specific role in breast tissue. The evidence that NB-1 expression is depressed in some primary breast tumor tissues and that NB-1 transcripts are undetectable in tumor-derived cell lines supports the possibility that loss of NB-1 gene expression is an important step in tumorigenic transformation of breast epithelial cells. We wish to express our thanks to Drs. Charles Dollbaum, Martha Dixon, Dieter Gruenert, and Kathy Zezulak for their gifts of cells and tissues; and to the Genentech Corporation for its gift of recombinant human TGFB1. This work was supported by National Institutes of Health Grant CA-24844 and the Office of Energy Research, Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC03-76SF00098 (M.R.S.), National Institutes of Health Grant CA 39814 (R.S.), and by the Lucas Foundation

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Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells.

A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease...
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