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[CANCER RESEARCH 53, 4096-Ã•I01. September 1, 1993]

Identification of a New Interferon-a-inducible Gene (p27) on Human Chromosome14q32 and Its Expression in Breast Carcinoma1

Ulla B. Rasmussen,2 Catherine Wolf, Marie-GeneviÃ¨ve Mattel, Marie-Pierre Chenard, Jean-Pierre Bellocq,Pierre ChambÃ³n, Marie-Christine Rio, and Paul Basset3

Laboratoire de CinÃ©tique MolÃ©culairedes Eucaryotes du CNRS, UnitÃ©184 de Biologie MolÃ©culaireet de GÃ©nieGÃ©nÃ©tiquede l'INSERM-lnstitut de Chimie Biologique-FacultÃ© deMÃ©decine,67085 Strasbourg Cedex, France Â¡U.B. R., C. W., P. C., M-C. R., P. B.J; UnitÃ©242 de Plnsiopalhologie Chromosomique de l'INSERM, HÃ´pital d'Enfants, GroupeHospitalier de la Timone, 13385 Marseille Cedex 5, France Â¡M-G.M,Â¡;and Service d'Anatomie Pathologique GÃ©nÃ©rale,Centre Hospitalier Universitaire-HÃ´pital de Hautepierre,

67098 Strasbourg Cedex, France /M-P. C., J-P. H./

ABSTRACT

A new complementary DNA, p27, has been cloned and sequenced fromestradiol-treated \1( I 7 human breast carcinoma cells. It encodes a puta

tive highly hydrophobic protein of 122 amino acids which has a 33%overall sequence similarity to the product of the 6-16 gene (R. L. Fried

man, S. P. Manly, M. McMahon, I. M. Kerr, and G. R. Stark, Cell, 38:745-755, 1984), which is transcriptionally induced by interfÃ©rons of theot/ÃŸtype. We demonstrate here that the /;_'/""gene, which is located in band

q32 of human chromosome 14, is also induced by interferon-a in human

cell lines of different origin and that expression is independent of thepresence of estradiol receptor in the cells. High levels of p27 RNA werefound in vim in approximately 50% of primary human breast carcinomas(21 were tested by Northern blotting). In situ hybridization to some of the/727-overexpressing tumors showed that the p27 RNA is localized in cancer

cells and sometimes also in fibroblastic cells of tumor stroma. p27 RNAlevels in the tumors did not correlate with the presence of estrogen receptor or with the expression of the estrogen-induced pS2 gene. Furtherstudies are now necessary to elucidate the cause of p27 gene overexpres-

sion in breast carcinoma and in particular to determine whether it corresponds to chromosomal rearrangements in the 14q32 region and/or toinduction by interferons of the a/ÃŸtype.

INTRODUCTION

In the industrialized world, with few exceptions, breast cancer is aleading cause of cancer deaths in women (1, 2). Early detection basedon mammography screening has improved the prognosis of the disease (3, 4), but it is clear that new treatments are necessary and thata prerequisite to the development of new therapeutic agents is a betterunderstanding of the disease at the molecular level (2).

Estrogens play an important part in the genesis of breast cancer(5, 6), and about one-third of the tumors require the presence of

estrogens for growing (7). In order to understand the molecular basisof estrogen action, several groups have screened for genes which areunder estrogen control in the ERJ-positive MCF7 breast cancer cell

line (8-10). Using the same strategy, we have identified a new genethat we termed p27 and which is overexpressed in estradiol-treated

MCF7 cells and in a number of breast carcinomas. p27 gene expression does not correlate with the presence of ER in human cell linesother than MCF7 nor in breast carcinoma. However, we found that the

Received 4/28/93: accepled 6/25/93.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore he hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by funds from the Centre National de la Recherche Scientifique, the

Institut National de la SantÃ©et de la Recherche MÃ©dicale,thÃ©Mutuelle GÃ©nÃ©raledel'Education Nationale, thÃ©Minislere de la Recherche et de l'Espace (contrat 92H.091), thÃ©

Centre Hospitalier Universitaire RÃ©gional,thÃ©Association pour la Recherche sur leCancer, thÃ©Ligue Nationale FranÃ§aisecontre le Cancer, thÃ©Fondation pour la RechercheMÃ©dicaleFranÃ§aise,thÃ©Groupement des Entreprises FranÃ§aisespour la Lutte contre leCancer, and thÃ©Fondation Jeantet.

: Present address: TransgÃ¨ne SA, 11, rue de Molsheim. 67082 Strasbourg, France.3 To whom requests for reprints should be addressed, at INSERM-U.184. Institut de

Chimie Biologique. 11. rue Humann. 67085 Strasbourg Cedex. France.4 The abbreviations used are: ER, estradiol receptor; IFN, Â¡nlerferon; DMEM.

Dulbccco's modified Eagle's medium: cDNA, complementary DNA; poly(A) *. poly-

adenylate.

p27 gene is induced by IFN-a in several human cell lines and that the

putative p27 protein has homology to the product of a previouslyidentified gene which is transcriptionally induced by IFNs of the a/ÃŸtype.

MATERIALS AND METHODS

Cell Lines and Tissue Culture. The human cell lines used in this studyincluded Ihe ER-positive (MCF7, BT-474, T-47D) and the ER-negative (HBL-100, SK-BR-3, MDA-MB-231 and BT-20) breast carcinoma cell lines, thegastric (KATO III), colon (Caco-2). and cervix (HeLa) carcinoma cell lines, thefetal diploid lung fibroblasts (HFL1). and the myeloid leukemia (HL-60 andK-562) cell lines. All of these cell lines are described in the American Type

Culture Collection catalogue (7th edition, 1992). Cells were maintained inDMEM supplemented with 10'i fetal calf serum and cultured until confluency

before harvesting for RNA preparation. Induction with estradiol was carriedout with MCF7 cells near confluency. Cells were washed with phosphate-buffered saline, refed with phenol red-free DMEM containing 10'? fetal calf

serum which had been stripped of endogenous steroid hormones by treatmentwith dextran-coated charcoal, and supplemented, or not. with 1 nMestradiol for3 days before harvesting. Induction with IFN-a was carried out with confluent

MCF7, HeLa, and HFL1 cells. Culture medium was removed and replaced byserum-free DMEM. After 24 h. fresh serum-free DMEM was added, .supplemented, or not, with IFN-a (0-1000 lU/ml, Roferon-A; Roche, Nutley, NJ),

and the incubation was continued for 24 h before harvesting.RNA Isolation and Northern Blots. Total RNA from cells in culture or

from surgical specimens (kept in liquid nitrogen until RNA extraction) wasextracted by guanidinium isothiocyanate solubilization and prepared by phenol-chloroform extraction (11) or cesium chloride gradient centrifugation (12).Total RNA (8 fig) was separated by electrophoresis in \r/r agarose gels con

taining formaldehyde and blotted onto Hybond-N filters (Amersham France,

Les Ulis, France). Hybridization and washing were performed under standardconditions, as described earlier (13). The probes used to hybridize the blotswere derived from full-length p27 cDNA (see "Results") and from the pS2 and

the 36B4 cDNAs (9). All probes were 32P-labeled by random primed DNA

synthesis and used successively on the same blots.Construction of cDNA Library and Probes for Differential Screening.

A subtracted cDNA library and the probes used for the differential screeningwere prepared according to the general procedure previously described (13). Inbrief, poly(A)+ RNA from MCF7 cells, cultured in the presence of estradiol.

was reverse transcribed and the single-stranded cDNA subtracted by using anexcess of poly(A)* RNA from the ER-negative MDA-MB-231 breast cancer

cell line according to the method of Rhyner et al. (14). After hydroxylapatitechromatography (15). the fractions enriched in cDNA sequences from theMCF7 cells were collected, and the cDNA was made double-stranded and

cloned into the Â£roRI site of a AgtlO vector. Part of the subtracted cDNA waskept single stranded and. after 12P-labeling, used as a positive probe to screen

the cDNA library. Differential screening was carried out by using a secondnegative probe, made as the first but starting with poly(A) ' RNA from MCF7

cells cultured in the absence of estradiol. Both probes were used to hybridizereplica filters from 12 plates, containing approximately 5000 phage plaqueseach. Hybridization conditions and treatment of the filters were as previouslydescribed (13).

Analysis of Selected Clones. After selection and a third screening, thecDNA inserts of the "differential plaques" were amplified by polymerase chain

reaction and used to probe replica filters and Northern blots, as describedbefore (16). For sequence analysis, the cDNA inserts were obtained by diges-

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p27 GENE EXPRESSION

tion of the purified recombinant phage DNA with the EcoRI restriction enzymeand subcloned into M13 vector, following standard procedures. Sequencingwas performed by the dideoxy-chain termination method, using the Sequenase/deaza-dGTP Reagent Kit (U. S. Biochemicals, Cleveland, OH). cDNA se

quences were analyzed with the DNASTAR software (DNASTAR. Madison,WI). and amino acid sequences were aligned using the method of Needlemanand Wunsch (17).

Gene Mapping. p27 gene mapping was carried out hy in situ hybridizationon chromosome preparations obtained from phytohemagglutinin-stimulatedhuman lymphocytes cultured for 72 h. 5-Bromodeoxyuridine (60 /ig/ml) was

added to the medium for the final 7 h of culture to ensure a posthybridizationchromosomal handing of good quality. The p27 cDNA, containing 598 basepairs cloned in the pBSII SK+ vector (Stratagcne, La Jolla, CA), was tritiumlabeled by nick-translation to a specific activity of 1.5 X 10" dpm/fxg. The

radiolabeled probe was hybridized to metaphase spreads at a final concentration of 25 ng/ml of hybridization solution, as previously described (18). Afterthe slides were coated with nuclear track emulsion (NTB2; Kodak, Rochester,NY), they were exposed for 19 days at 4Â°Cbefore development. To avoid any

slipping of silver grains during the banding procedure, chromosome spreadswere first stained with buffered Giemsa solution, and metaphases were photographed. R-banding was then performed by the fluorochrome-photolysis-

Giemsa method, and metaphases were rephotographed before analysis.RNA in Situ Hybridization. In situ hybridization was performed on form

aldehyde-fixed paraffin-embedded tissue sections as previously described (13).In brief, 8-fim-thick rehydrated and acid-treated sections were digested withproteinase K (2 fig/ml-30 min, 37Â°C), followed by overnight hybridization(0.3 M NaCl-50% formamide, 50Â°C)with a 15S-labeled p27 antisense RNA

probe (specific activity, W cpm/^g). The sections were RNase treated (20jj.g/ml, 30 min, 37Â°C)and stringently washed (2X standard sodium citrate-50% formamide, 60Â°C,twice for 2 h). p27 antisense RNA was obtained by in

vitro transcription of the full-length p27 cDNA from the pBSII SK+ vector.

Autoradiography was for 3 to 4 weeks using the NTB2 emulsion. After exposure, the slides were developed and stained with hematoxylin. The ER statusof tumors was determined by immunohistochemical analysis as previouslydescribed (19).

RESULTS

Cloning of the p27 cDNA. A total of 60,000 plaques from asubtracted MCF7/estradiol-positive cDNA library was differentially

screened using one probe derived from MCF7 cells cultured in thepresence of estradiol [MCF7/estradiol-positive] and a second probe

derived from MCF7 cells cultured in the absence of estradiol [MCF7/estradiol-negative]. A clearly higher hybridization signal with theMCF7/estradiol-positive than with the MCF7/estradiol-negative probewas given by 318 plaques, and 252 of these "differential clones"

corresponded to the estradiol-inducible pS2 gene (9). In a second

screening of the remaining clones, 21 showed a differential signal, but15 corresponded, again, to pS2. After a third screening, 4 clones stillshowed a differential signal, and their inserts were then used to probeNorthern blots prepared with MCF7/estradiol-positive and MCF7/estradiol-negative RNAs. Three of them hybridized to a 0.6-kilobase

RNA band which was present at high levels only on the MCF7/estradiol-positive blot (Fig. 1, lanes 1 and 2). The fourth insert did not

hybridize to any RNA (data not shown). The 3 cDNAs that hybridizedto the 0.6-kilobase estradiol-induced transcripts were sequenced and

found to correspond to the same gene that we termed p27 (Fig. 2).Characterization of the p27 cDNA. p27 RNA is the same size as

pS2 RNA (9, 20), and it Â¡salso similarly induced by estradiol in theMCF7 cells (Fig. 1, lanes 1 and 2). However, the p27 cDNA sequenceindicates that it corresponds to an entirely different gene (Fig. 2) andthat it contains an open reading frame which codes for a putativeprotein of 122 amino acids with a calculated molecular weight of11,528. The putative initiation codon (nucleotides 55â€”57)is found in

a typical eukaryotic initiation consensus sequence (21), and a singlepolyadenylation signal is seen 21 nucleotides upstream from the start

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12 345678 9 10 11 121314Fig. 1. Comparative Northern blot analysis of p27 and pS2 RNAs in human cell lines.

Northern blots were prepared with 8 jag of total RNA from cells at confluency, in eachsample. Filters were successively hybridized with 32P-labeled human cDNAs corresponding lo the p27 gene, the pS2 gene [an estradiol-inducible gene (9)], and the 36B4 gene [anubiquitously expressed gene (19)]. p27 and pS2 transcripts were detected at 0.6 kitobases,and 36B4 transcripts were detected at 1.5 kilobases. Autoradiography was for 16 h.

of a possible poly(A) tail. A hydropathicity plot (not shown) indicatesthat the predicted p27 protein is highly hydrophobic and, therefore,possibly corresponds to a membrane protein, but it contains no typicalsignal peptide sequence at the NH2 terminus.

A database search (National Biomedicai Research Foundation-Pro

tein Identification Resource, Washington, DC, 1992, release 32)showed that the p27 protein has highest amino acid similarity (41%identity in a 116 amino acid overlap, 33% overall) to a 130-amino acid

protein which has been predicted from the sequence of a cDNA clonedfrom T98G neuroblastoma cells (22), and termed 6-16 (Fig. 2B). The6-76 gene is induced by IFN-a/ÃŸin a variety of human cells, includ

ing T98G neuroblastoma cells and HeLa cells (22, 23). The functionof the putative 6-16 protein is unknown, but it is suggested to be amembrane protein, with a highly charged carboxy-terminal tail and a

typical signal peptide sequence at the NH2 terminus, both apparentlyabsent in the putative p27 protein.

p27 Gene Localization to Chromosome 14q32. In situ hybridization on human chromosome preparations was carried out using â€¢¿�1H-

labeled p27 cDNA. In the 200 metaphase cells examined after in situhybridization, there were 564 silver grains associated with chromosomes, and 69 of these (12.2%) were located on chromosome 14. Thedistribution of grains on this chromosome was not random: 49 of 69(71%) mapped to the q31-q32 region of chromosome 14 long arm

with a maximum in the q32 band (Fig. 3). Thus, the p27 gene islocalized on the 14q32 band of the human genome, which is frequently involved in chromosomal translocations associated with lym-

phoid malignancies (24, 25).Â¡>27Gene Expression in Human Cell Lines and Its Induction by

Interferon-a. A number of human cell lines, cultured in the presenceof estradiol-containing fetal calf serum, were analyzed for p27 gene

expression by Northern blotting. In contrast to pS2 RNA, p27 RNAwas not detected in the ER(+) BT-474 cells (Fig. 1, lane 3), and verylow levels of p27 RNA were seen in the ER(+) T-47D cell line (Fig.

1, lane 4). However, high levels of p27 transcripts were seen in theER-negative HBL-100 (Fig. 1, lane 5) and SK-BR-3 (Fig. 1, lane 6)

cells. The p27 gene was not expressed in the other human epithelialand nonepithelial cell lines which were tested (Fig. 1, lanes 7, 8, and10-14) with the exception of KATO III (Fig. 1, lane 9), a cell linederived from an ER-negative gastric carcinoma.

Altogether, these observations indicate that p27 gene expressiondoes not correlate with ER expression in human cell lines, although itis induced by estradiol in the MCF7 cells. Since the putative p27protein exhibited 41% identity with the 6-16 protein, for which theexpression is induced by IFN-a/ÃŸ (22, 23), we tested whether p27

4097



p27 GENE EXPRESSION

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Fig. 2. NucletÃ¬tidcsequence of p27 cDNA and comparison of the amino acid sequences of p27 and 6-16 putative proteins. A, nucleotidc sequence of p27 cDNA (accession no.X67325 in the EMBL. GenBank. and DDBJ nucleotidc sequence data bases) and deduced amino acid sequence. Amino acids are represented wilh the one-letter code. A polyadcnvlationsignal sequence is underlined, fl. comparison of the predicted amino acid (one-letter code) sequences of p27 and 6-16 proteins (23). Amino acid residues were aligned using the methodof Needleman el al. (17). Gaps in the 6-16 sequence (-) were introduced to obtain maximal similarity. Identical amino acids are shown between the two sequences, and dois denote

conservative amino acid changes.

gene expression is induced by IFN-a. IFN-a was indeed found to

stimulate p27 gene expression in the 3 cell lines tested, of eitherepithelial (MCF7 and HeLa cells) or mesenchymal (HFL1 fibroblasts)origin (Fig. 4), which do not constitutively express the gene (the lowlevel of p27 RNA in MCF7 cells before IFN-a addition was most

probably due to the presence of residual levels of estradiol in theculture, as indicated by the simultaneous presence of pS2 RNA). p27RNA was induced in a dose-dependent manner by IFN-a, and theeffect was specific for the p27 gene since IFN-a did not increase pS2

or 36B4 gene expression in the same cells (Fig. 4).p27 Gene Expression in Breast Carcinoma. p27 RNA expression

was evaluated by Northern blot analysis of 21 primary invasive breastcarcinomas, 1 breast cancer bone metastasis, and 3 breast fibroade-

nomas. High levels of p27 transcripts, by comparison with thoseobserved in the 3 fibroadenomas, were found in approximately 50% ofthe primary breast carcinomas tested and in the bone metastasis (Fig.5A and data not shown). Furthermore, p27 gene overexprcssion wasnot correlated with ER expression in the tumors (for instance, ER wasnot immunodetected in carcinoma 5 or 7, but 90% of cancer cells wereER( + ) in carcinoma 8; Fig. 5A and data not shown), and the patternof p27 gene expression differed from that of pS2 (Fig. 5A ) which is aknown marker of ER-dependent breast carcinomas (19, 26). The p27gene was also expressed at significant levels in some normal non-

breast tissues, including colon, stomach, and lung (Fig. 5ÃŸ).Seventeen primary invasive breast carcinomas, 4 breast cancer

metastatic lymph nodes, 1 breast cancer bone metastasis, and 44098



p27 GENE EXPRESSION

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Fig. 3. Localization of p27 gene on chromosome 14q32. Top and middle, 2 partialhuman metaphases showing the specific site of hybridization to chromosome 14. Top.arrowheads, silver grains on Giemsa-stained chromosomes after autoradiography. Middle,chromosomes with silver grains subsequently identified by R-banding (fluorochrome-photolysis-Giemsa technique). Bottom, idiogram of the human G-banded chromosome 14illustrating the distribution of labeled sites for the p27 cDNA probe.

breast fibroadenomas were analyzed by in situ hybridization for p27gene expression. p27 transcripts were not detected at significant levels in the normal cells of the tissue sections (Fig. 6, a and b, anddata not shown), but a low level of p27 RNA was observed in fibro-

blasts of 2 fibroadenomas (data not shown). In contrast, high levelsof p27 transcripts were detected in cancer cells of 13 of the primarybreast carcinomas and 4 of the mÃ©tastases(one metastatic lymphnode was negative) (Fig. 6, a-d, g, and h, and data not shown). In

the primary tumors, both invasive (Fig. 6, a and b) and in situ (Fig.6, c and d) cancer cells expressed the p27 gene. p27 gene expressionwas also observed in stromal cells of 4 primary tumors and 2 mÃ©tastases (Fig. 6, e and / and data not shown). Furthermore, cancer cellsthat were surrounded by p27-expressing stromal cells did not express

the p27 gene (Fig. 6, e and /), but they did in other parts of the same

tumor where the gene was not expressed in the stromal cells (Fig. 6,a-d). We found no p27 gene expression in inflammatory cells of the

tumor stroma, and the levels of p27 transcripts did not appear to becorrelated with the intensity of the inflammatory component of tumors (data not shown).

DISCUSSION

We have identified a new gene, p27, which maps to position14q32 on the human genome. The p27 cDNA contains an open reading frame which codes for a putative 122-amino acid highly hydro-

phobic protein, suggesting that it is a membrane protein. The proteinsequence shows 33% overall similarity to a previously reported putative protein which is encoded by the 6-76 gene (22, 23). Although

expression of the p27 gene is induced by estradiol in MCF7 breastcancer cells, it is not dependent on the presence of ER in other celllines, nor in primary breast carcinomas where p27 gene expressiondoes not correlate with that of the estrogen-induced pS2 gene. Evidently, high levels of p27 transcripts were detected both in ER-negative cell lines and in ER-negative breast carcinomas. After noting the homology on the protein level between p27 and 6-16, whichis highly induced by IFN-a/ÃŸ in a variety of human cell lines (22,23), we tested and found that the p27 gene is also induced by IFN-a

in cells of different origin.The IFNs, which can be classified as cytokines, are part of the

body's natural defense responses to tumors, and it has been sug

gested that the IFN-a/ÃŸgenes represent a class of tumor suppressorgenes (27-30). They exert antitumor effects both by affecting the

functioning of the immune system and by direct action on certain tumor cells (31). IFNs are believed to mediate these multiple effectsby inducing or repressing the synthesis of several proteins (32-34),

and the p27 protein may belong to this group. Whether p27 gene expression is controlled by IFNs in vivo is unknown, but it is not unlikely, since most cell types appear capable of producing and bindingIFN-a/ÃŸ (32, 34). Furthermore, p27 RNA was present both in the

neoplastic cells and in the fibroblastic cells of breast carcinomas, anobservation which is consistent with the in vitro results, showing p27gene induction by IFN-a in cell lines of both epithelial and mesen-

chymal origin. That increased p27 gene expression was observed inonly some breast tumors may indicate that these tumors producehigher levels of, or have higher sensitivity to, IFN-a/ÃŸthan others.

Alternatively, increased p27 gene expression in breast carcinomamay be a result of chromosomal rearrangements in the 14q32 region.In this respect, we note that the 14q32.l locus, to which the p27

MCF7 HeLa HFL1

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1234 5 6 7 8 9 10 11 12Fig. 4. Induction of p27 RNA by interferon-a in human cell lines. Confluent MCF7,

HeLa, and HFL1 cells were stimulated with increasing concentration of interferon-a(0-1000 lU/ml), as described in "Materials and Methods." After 24 h of treatment, cells

were harvested and used to prepare RNA. Northern blot analysis was carried out asdescribed in the legend to Fig. 1.

4099



Fig. 5. Comparative Northern blot analysis ofp27 and pS2 RNAs in human tissues. A, breasttumors: lunes 1-10, invasive carcinomas; lane 11,

bone metastasis; lanes 12 and Â¡3.fibroadenomas.H, normal tissues. Northern blot analysis was carried out as described in the legend to Fig. 1.

p27 GENE EXPRESSION

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Fig. 6. In situ hybridization of p27 RNA on breast cancer tissue sections, a, c, e, and g, bright-field photomicrographs of paraffin-embedded tissue sections (X KH1)stained withhematoxylin; b, d, f, and /i, dark-field photomicrographs of the same sections in which p27 transcripts appear as white silver precipitates after /'// situ hybridization with ^S-labeled

p27 antisense RNA. aâ€”f.3 distinct areas of the same tumor: a and b. infiltrating ductal carcinoma in which p27 transcripts are detected in neoplastic cells (C) but not in stromal cells(S) or in normal epithelial cells (N); c and d. 2 ducts containing ductal in situ carcinomas (*), one of which expresses the p27 gene; e and f. ductal infiltrating carcinoma in which

p27 transcripts are detected in stromal cells (S) but not in neoplastic cells (C). g and h. breast cancer metastatic lymph node expressing the p27 gene.

gene belongs, contains a putative oncogene (24). However, it is unlikely that chromosomal rearrangements could be involved in p27gene overexpression when it is observed in the stromal componentof tumors.

Further studies are now required to understand the meaning of p27gene overexpression in breast carcinoma and to determine whether thelevels of p27 can be used to identify subgroups of tumors with specificbiological and clinical characteristics. In this respect, it will be important to determine the cause(s) of p27gene overexpression in breastcarcinoma.

ACKNOWLEDGMENTS

We thank R. Ceredig for critically reading the manuscript, H. V. J. Kolhc lorsuggestions during the work, I. Stoll, C. Wendung, and M. Loriot for technicalassistance, and the Michigan Cancer Foundation for MCF7 breast cancer cells.

REFERENCES

1. Davis. D. L.. Hoel, D.. Fox, J., and Lopez, A. International trends in cancer mortalityin France. West Germany. Italy. Japan. England and Wales, and the USA. Lancet, 336:474-4X1. 1<W().

2. Harris, J. R., Lippman. M. E., Veronesi. U., and Willen. W. Breast Cancer. N. Engl.

4 KM)



p27 GENE EXPRESSION

J. Med., 527: 319-328. 390-398. 473-480, 1990.

3. Forrest, A. P. M., and Aitken, R. J. Mammography screening for breast cancer. Annu.Rev. Med., 41: 117-132, 1990.

4. Wald. N.. Frost, C., and Cuckle, H. Breast cancer screening: the current position. Br.Med. J., 302: 845-846, 1991.

5. Henderson, B. E., Ross, R., and Bernstein, L. Estrogens as a cause of human cancer:the Richard and Hinda Rosenlhal Foundation Award lecture. Cancer Res., 48: 246-253, 1988.

6. Lippman. M. E., and Dickson, R. B. Mechanisms of growth control in normal andmalignant breast epithelium. Recent Prog. Horm. Res., 45: 383^(40, 1990.

7. Jensen, E. V, Greene, G. L., Closs, L. E., DeSombre, E. R., and Nadji, M. Receptorsconsidered: a 20 year perspective. Recent Prog. Horm. Res., 38: l-W, 1982.

8. Westley. B., and Rochefort, H. A secreted glycoprotein induced by estrogen in humanbreast cancer cell lines. Cell. 20: 352-362, 1980.

9. Masiakowski, P., Breathnach, R., Bloch, J., Gannon, F., Krust, A., and ChambÃ³n, P.Cloning of a cDNA sequence of hormone-regulated genes from the MCF-7 humanbreast cancer cell line. Nucleic Acids Res., 10: 7895-7903, 1982.

10. May, F. E. B., and Westley, B. R. Identification and characterization of estrogen-regulated RNAs in human breast cancer cells. J. Biol. Chem., 263: 12901-12908,1988.

11. Chomczynski. P.. and Sacchi, N. Single-step method of RNA isolation by acidguanidinium thiocyanate phenol chloroform extraction. Anal. Biochem., 762: 156-

159. 1987.12. Chirgwin, J. M.. Przybyla, A. E., MacDonald, R. J., and Rutter. W. J. Isolation of

biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 18: 5294-5299, 1979.

13. Basset, P., Bellocq, J. P., Wolf, C, Stoll, I., Hutin, P., Limacher, J. M., Podhajcer. O.L., Chenard, M. P., Rio. M. C. and ChambÃ³n. P. A novel metalloproleinase genespecifically expressed in stromal cells of breast carcinoma. Nature (Lond.), 348:699-704, 1990.

14. Rhyner, T. A., Faucon Biguet, N., Berrard, S., Borbely, A. A., and Mallet, J. Anefficient approach for the selective isolation of specific transcripts from complex brainmRNA populations. J. Neurosa. Res., 16: 167-181, 1986.

15. Hedrick, S. M., Cohen, D. I., Nielsen. E. A., and Davis, M. M. Isolation of cDNAclones encoding T-cell specific membrane associated proteins. Nature (Lond.), 308:149-153, 1984.

16. Rasmussen, U. B., Basset, P., and Daniel. J. Y. Direct amplification of cDNA insertsfrom lambda libraries using the cloning adaptor as primer for PCR. Nucleic AcidsRes., 17: 3308. 1989.

17. Needleman, S. B., and Wunsch, C. D. A general method applicable to the search forsimilarities in the amino acid sequence of two proteins. J. Mol. Biol., 48: 443^153,1970.

18. Mallei, M. G., Philip, N., Passage, E., Moisan, J. P., Mandel, J. L., and Mattei, J. F.DNA probe localization at I8pll.3 band by in situ hybridization and identification of

a small supernumerary chromosome. Hum. Genet., 69: 268-271, 1985.

19. Rio, M. C., Bellocq, J. P.. Gairard, B., Rasmussen, U. B., Krust, A., Koehl, C.,Calderoli, H., Schiff, V., Renaud, R., and ChambÃ³n, P. Specific expression of the pS2gene in subclasses of breast cancers in comparison with expression of the estrogen andprogesterone receptors and the oncogene ERBB2. Proc. Nati. Acad. Sci. USA, 84:9243-9247. 1987.

20. Jakowlew, S. B., Breathnach. R.. Jeltsch, J. M., Masiakowski, P., and ChambÃ³n, P.Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell lineMCF7. Nucleic Acids Res., 12: 2861-2878, 1984.

21. Kozak, M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger

RNAs. Nucleic Acids Res.. 15: 8125-8148, 1987.22. Friedman, R. L., Manly. S. P., McMahon, M., Kerr, I. M., and Stark, G. R. Tran-

scriptional and post-transcriptional regulation of interferon-induced gene expressionin human cells. Cell, 38: 745-755, 1984.

23. Kelly, J. M., Porter, A. C. G.. Chernajovsky, Y, Gilbert, C. S., Stark, G. R., and Kerr,I. M. Characterization of a human gene inducible by a- and ÃŸ-interferons and itsexpression in mouse cells. EMBO J., 5: 1601-1606, 1986.

24. Russo, G., Isobe, M., Gatti, R., Finan, J., Batuman, O., Huebner, K., Nowell, P. C.,and Croce, C. M. Molecular analysis of a t(14; 14) translocation in leukemic T-cellsof an ataxia telangiectasia patient. Proc. Nati. Acad. Sci. USA, 86: 602-606, 1989.

25. Mitelman, F., Kaneko, Y, and Trent, J. Report of the comittee on chromosomechanges in neoplasia. Cytogenet. Cell Genet., 58: 1053-1079, 1991.

26. Foekens, J. A., Rio, M. C., Seguin, P.. van Putten, W. L. J., Fauque, J., Nap, M., Klijn,J. G. M., and ChambÃ³n, P. Prediction of relapse and survival in breast cancer patientsby pS2 protein status. Cancer Res., 50: 3832-3837, 1990.

27. Cowan, J. M.. Halaban, R.. and Francke, U. Cytogenetic analysis of melanocytes frompremalignant nevi and melanomas. J. Nati. Cancer Inst., 80: 1159-1164, 1988.

28. Diaz, M. O., Ziemin. S.. LeBeau, M. M., Pitha, P.. Smith, S. D., Chilcote, R. R., andRowley, J. D. Homozygous deletion of the a- and ÃŸi-interferon genes in humanleukemia and derived cell lines. Proc. Nati. Acad. Sci. USA, 85: 5259-5263, 1988.

29. Pitha, P. M. Interferons: a new class of tumor suppressor genes? Cancer Cells (ColdSpring Harbor), 2: 215-216, 1990.

30. James, C. D., He, J., Carlbom, E., Nordenskjold. M., Cavenee, W. K., and Collins, V.P. Chromosome 9 deletion mapping reveals interferon a and interferon ÃŸlgenedeletions in human glial tumors. Cancer Res., 51: 1684-1688, 1991.

31. Baron, S., Tyring, S. K., Fleischmann, W. R., Coppenhaver, D. H., Niesei, D. W.,Kumpel. G. R.. Stanton. G. J., and Hughes, T. K. The interferons: mechanisms ofaction and clinical applications. JAMA, 266: 1375-1383, 1991.

32. Pestka, S., Langer, J. A., Zoon, K. C., and Samuel, C. E. Interferons and their actions.Annu. Rev. Biochem., 56: 727-777, 1987.

33. Kerr, I. M., and Stark, G. R. The control of interferon-inducible gene expression.FEES Lett., 285: 194-198, 1991.

34. Sen, G. C., and Lengyel, P. The interferon system. J. Biol. Chem., 267: 5017-5020,1992.

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1993;53:4096-4101. Cancer Res Ulla B. Rasmussen, Catherine Wolf, Marie-Geneviève Mattei, et al. CarcinomaHuman Chromosome 14q32 and Its Expression in Breast

) onp27-inducible Gene (αIdentification of a New Interferon-

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