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[Cancer Research 60, 5323-5328, October 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

MYB Oncogene Amplification in Hereditary BRCA1 Breast Cancer1

Päivikki Kauraniemi2, Ingrid Hedenfalk2, Karin Persson, David J. Duggan, Minna Tanner, Oskar Johannsson, Håkan Olsson, Jeffrey M. Trent, Jorma Isola and Åke Borg3

Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, FIN-33101 Tampere, Finland [P. K., M. T., J. I.]; Department of Oncology, University Hospital, SE-22185 Lund, Sweden [I. H., K. P., M. T., O. J., H. O., J. I., A. B.]; Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892 [I. H., D. J. D., J. M. T.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Comparative genomic hybridization analysis has demonstrated that breast tumors from BRCA1 and BRCA2 germ-line mutation carriers contain a large number of chromosomal copy number gains and losses. A high regional copy number gain at 6q22-q24 was observed in one BRCA1 tumor, and fluorescence in situ hybridization analysis indicated a strong amplification of the MYB oncogene (15 copies of MYB compared with 1 copy of chromosome 6 centromere). Fluorescence in situ hybridization analysis revealed amplification of MYB in 5 (29%) of 17 BRCA1 breast tumors, whereas none of 8 BRCA2 tumors and 13 breast cancer cell lines, and only 2 of 100 sporadic breast tumors exhibited altered MYB copy numbers. Gene amplification resulted in mRNA overexpression as determined by Northern blot and cDNA microarray analysis, and protein overexpression by immunohistochemical staining. We conclude that MYB amplification is infrequent in sporadic breast cancer but common in breast tumors from BRCA1 mutation carriers, suggesting a role of this cell cycle regulator and transcription factor in the progression of some BRCA1 tumors. However, we cannot rule out the significance of other genes in the 6q22-q24 amplicon.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Breast tumors from BRCA1 and BRCA2 mutation carriers are characterized by a large number of chromosomal copy number gains and losses, as seen by CGH4 (1) . The apparent genomic instability in these tumors is consistent with the proposed role of BRCA proteins in recombination-mediated double-strand DNA break repair, transcription-coupled repair, centrosome amplification control, and G2-M cell cycle checkpoint (reviewed in Ref. 2 ). CGH analysis has also demonstrated that BRCA1- and BRCA2-associated tumors display specific patterns of genetic changes, clearly distinguished from sporadic breast cancer (1) . This suggests that tumor evolution progresses along distinct pathways in these genetically predisposed individuals. Indeed, inactivation of the p53 checkpoint function has been proposed as a prerequisite for development of these tumors (3) . Similarly, the high frequency of 4q and 5q deletions in BRCA1 tumors, as well as 17q23 and 20q gain in BRCA2 tumors, may signal the sites of other genes with selective roles in keeping or overriding cell cycle control and genome integrity (1) . Gene amplification is a common mechanism of oncogene activation in solid tumors such as breast cancer. Amplified DNA typically retained in autonomously replicating extrachromosomal bodies may rapidly increase in number in an evolving cell clone by unequal distribution at mitosis, or be successively lost if not providing a growth advantage. CGH analysis of fresh-frozen breast tumor tissue from BRCA1 mutation carriers, identified through genetic testing of Scandinavian breast cancer families in Lund, Sweden, demonstrated an exceptionally distinct copy number gain at chromosome 6q22-q24 in one tumor. This is a novel amplified region in breast cancer and justified further studies in hereditary and sporadic breast tumors, as well as attempts to identify the putative target gene(s) of the amplicon.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Tumors and Cell Lines.
Primary tumors were received from pathology departments in the southern Sweden health care region and stored freshly frozen at -70°C. The present study included 17 tumors from BRCA1 mutation carriers, 8 from BRCA2 mutation carriers, and 100 sporadic breast tumors. BRCA1 and BRCA2 mutation analyses have been described earlier (4) , and consisted of the protein truncation test, single-strand conformational polymorphism analysis, and direct sequencing. Thirteen established breast cancer cell lines (BT-474, DU4475, MDA134, MDA157, MDA361, MDA436, MCF-7, MPE600, SKBR3, T47D, UACC812, UACC893, and ZR75-1) were grown in recommended conditions and harvested at confluency to obtain interphase nuclei from cells that were predominantly in the G1-phase of the cell cycle (5) . Trypsinized cells were cytocentrifuged and air-dried at room temperature and fixed in Carnoy’s fluid [methanol:acetic acid (3:1)].

Probes for FISH.
PAC probes for MYB, MYBL1 (A-MYB), MYBL2 (B-MYB), and the ER{alpha} gene were obtained by screening a PAC library by PCR using primers specific for each gene. The specificity of the probes was confirmed by FISH to normal metaphase chromosomes, which showed the presumed chromosomal localization for each probe. The probes were labeled with digoxigenin by standard nick translation. A spectrum green-labeled pericentromeric probe (Vysis, Inc., Downers Grove, IL) was used as a reference probe to determine the copy number of chromosomes 6, 8, and 20 for MYB, MYBL1, and MYBL2, respectively.

FISH.
Touch imprint preparations were made for FISH analysis by lightly pressing a semi-thawed frozen tumor onto Superfrost Plus microscope slides (Menzel, Braunschweig, Germany) microscope slides and air-dried. Prior to hybridization, imprint preparations were fixed with 50, 70, and 100% Carnoy’s solution [methanol:acetic acid (3:1)] for 10 min each. Dual-color FISH experiments were performed as described previously (5) . MYB, MYBL1, MYBL2, and ER probes were hybridized together with chromosome 6 centromere probe. The hybridization was carried out overnight at 42°C in a mixture containing 5 ng of pericentromeric probes, 20 ng of gene-specific probes, and 10 µg of human placental DNA. After hybridization, excess probes were washed with 0.4x SSC (2 min at 74°C) and 2x SSC (1 min at room temperature), and detected immunohistochemically with antidigoxigenin rhodamine. Slides were counterstained with 0.2 mM 4,6-diamidino-2-phenylindole in an antifade solution (Vectashield; Vector Laboratories, Burlingame, CA). Hybridization signals were evaluated using an Olympus BX50 epifluorescence microscope equipped with a x63 oil-immersion objective (numeric aperture, 1.4). A dual band-pass fluorescence filter (Chromotechnology; Brattleboro, VT) was used to visualize the FITC and rhodamine signals simultaneously.

At least 80 nonoverlapping nuclei with intact morphology based on 4,6-diamidino-2-phenylindole counterstaining were scored to determine the number of hybridization signals for MYB and centromere probes. Control hybridizations to normal lymphocytes were done to ascertain that the probes recognized a single copy target and that the hybridization efficiencies were sufficient. Both absolute copy numbers and the copy number ratio (between average of MYB, MYBL1, and MYBL2 and the respective centromere copy numbers) were determined. Amplification of MYBgene was defined as a copy number ratio of >=2.0, whereas a copy number gain was assigned to tumors having a ratio >1.5 and <2.0.

CGH.
CGH was performed according to a published protocol (6) . Briefly, tumor DNA and normal female reference DNA was extracted using a standard protocol and labeled with FITC-dCTP and Texas-Red-dUTP (DuPont, Boston, MA), respectively, using standard nick translation. Labeled DNAs (400–800 ng of each) and 10 µg of unlabeled Cot-1 DNA (Life Technologies, Gaithersburg, MD) were hybridized onto commercially available normal metaphase chromosomes (Vysis). The hybridizations were evaluated by a commercial digital image analysis system (Vysis).

Northern Blot.
Total RNA was extracted from the tumors with the RNeasy kit (Qiagen), followed by TRIzol (Life Technologies). Ten µg of RNA from each tumor was size-fractionated on a 1% agarose gel containing formaldehyde. After capillary transfer to Hybond-N membranes, the RNA was hybridized with a 32P-labeled MYB probe. Following hybridization overnight at 42°C, the membranes were washed twice in 2x SSC containing 0.1% SDS at 42°C and twice in 0.1x SSC containing 0.5% SDS at 65°C before exposure to autoradiographic film at -70°C.

cDNA Microarray Preparation, Hybridization, and Analysis.
Microarrays were prepared by PCR amplification and arrayed on poly-L-lysine-coated glass slides using a custom, high-speed robotic printer as described previously (7) . Total RNA was extracted from the biopsy specimen and the reference MCF-10A cells using RNeasy (Qiagen) and TRIzol in succession according to the manufacturers’ recommendations. Fluorescent-labeled cDNA targets were prepared by a single round of reverse transcription in the presence of Cye3- or Cye5-dUTP (Amersham) and hybridized to the probes as described previously (8) . Fluorescence intensities were measured using a custom-designed scanning laser confocal microscope with appropriate excitation and emission filters, and a photomultiplier tube. ArraySuite software (9) was then used to identify probe sites, extract fluorescence intensities, and merge the two images. Color images were formed by arbitrarily assigning the red (Cye3) and green (Cye5) channels. Local background was calculated for each probe location. The ratios for all of the probes and confidence intervals for each experiment were determined with the aid of 88 "housekeeping" genes whose theoretical expression ratios are expected not to deviate significantly from 1.0 (7 , 9) .

Immunohistochemistry.
A tumor xenograft from tumor 13996 was grown in nude mice as described elsewhere.5 Freshly prepared xenograft tissue was fixed overnight in 4% buffered formalin and processed to a paraffin block according to a standard procedure. Sections (3–4 µm) were cut onto poly-L-lysine-coated slides. Prior to immunostaining, antigen retrieval was performed by immersing the dewaxed sections in 10 mM EDTA (pH 8.0) at 94°C for 20 min in a temperature-controlled microwave oven. A standard avidin-biotin-peroxidase technique was used for visualization with diaminobenzidine as a chromogen (Histostain Plus-kit; Zymed Laboratories, San Francisco, CA). A polyclonal antibody to c-Myb was obtained from Santa Cruz Technologies and was used in a 1:500 dilution.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Identification of MYB Amplification.
Analysis of relative copy number gains and losses by CGH indicated that tumor 13996 from a germ-line BRCA1 mutation (1806C->T) carrier showed a high-level copy number gain at 6q22-q24 (Fig. 1A)Citation . A search in the gene bank and a recent study of pancreatic carcinoma (10) suggested MYBoncogene as a possible target gene for the amplification. A large-insert size PAC probe was prepared for MYB and used in FISH. A high-level amplification was verified in the imprint touch preparation of the same tumor (mean of 15 copies of MYB and 1 copy of chromosome 6 centromere; Fig. 1BCitation ). As could be estimated from CGH, the amplicon is small and did not include the ER{alpha} gene, located at 6q25 (FISH data not shown).



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  		Fig. 1. A, comparative genomic hybridization showing a high-level copy number gain at 6q22–24 in a primary breast cancer (13996) from a germ-line BRCA1 mutation carrier. The pseudocolored image of chromosome 6 is shown on the left; the corresponding green-to-red fluorescence ratio profile is shown on the right. B, FISH demonstrating high-level amplification of MYB in a BRCA1 tumor (13996). C, FISH of a BRCA1tumor (14510) with low-level amplification of MYB. D, FISH of a BRCA1 tumor (12224) with no MYB amplification. Note that not all amplified gene copies (B–D, in red) are in the focal plane. Chromosome 6 centromere is shown in green fluorescence.

 
Amplification of MYB in Breast Tumors by FISH.
FISH analysis of 17 tumors from 16 BRCA1 germ-line mutation carriers showed that MYB amplification is common in this tumor entity. Amplification was found in 5 (29%) of the 17 tumors, with a MYB/centromere copy number ratio ranging from 2.27 to 11.2. The mean MYB gene copy number in amplified tumors varied between 6.6 and 21.2, reflecting a high degree of aneuploidy in some tumors (Table 1)Citation . One tumor showed a borderline copy number gain (mean, 10.5 copies of MYB versus 5.8 copies of chromosome 6 centromere, giving a ratio of 1.80). Highest amplification was seen in tumor 13996. Interestingly, a bilateral tumor (8571) from this patient had no MYB amplification (Table 1)Citation . MYB amplification was found in none of the 8 tumors from BRCA2 mutation carriers, in 2 of 100 sporadic breast cancers, and in none of the 13 breast cancer cell lines. The ratio of MYB/chromosome 6 centromere copy number in the two amplified sporadic tumors was in the same range as in amplified BRCA1 tumors (5–10; data not shown). Examples of tumors with a moderate level of MYB amplification and no MYB amplification are shown in Fig. 1, C and DCitation , respectively. No evidence of MYBL1(A-MYB) or MYBL2 (B-MYB) gene amplification was seen in the present series of BRCA1, BRCA2, and sporadic breast tumors (data not shown).


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  		Table 1 MYB copy numbers in primary breast cancers from germ-line BRCA1 and BRACA2 mutation carriers

 
Overexpression of MYB Transcripts, and Correlations to Hormone Receptor Status.
Northern blot analysis showed that BRCA1 tumors (13996-xeno, 12421, 8822, and 14510) with MYB gene amplification manifested elevated MYB expression compared with tumors without MYB amplification (Fig. 2)Citation . Tissue for RNA extraction was not available from the remaining amplified tumors (BRCA1 tumor 10359 and two sporadic breast tumors). Two of five BRCA1 tumors without MYB gene amplification manifested MYB overexpression, whereas the remaining three showed low, borderline, and absent MYB expression, respectively. BRCA1 tumors were hormone receptor-negative, the only exception being tumor 12421, which showed borderline ER expression (12 fmol/mg of protein). Five sporadic tumors without MYB amplification where analyzed by Northern blot. MYB expression was seen in the three hormone receptor-positive sporadic tumors, whereas MYB transcripts were absent in two ER/PgR-negative tumors. All BRCA2 tumors were positive (>10 fmol/mg of protein) or strongly positive (>200 fmol/mg of protein) for either ER or PgR and manifested varying levels of MYB expression, two tumors being of borderline positivity.



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  		Fig. 2. Northern blot analysis of MYB mRNA expression in BRCA1, BRCA2, and sporadic breast cancer. The same membranes were hybridized with an actin probe, and based on densitometry evaluation of MYB/actin band intensity ratios, MYB expression was scored as negative (-), borderline [(+)], or positive (+, + +, + + +, + + + +, + + + + +). MYB gene copy number was scored according to Table 1Citation as normal (-), amplified (+), or highly amplified (+ +). ER and PgR expression was scored as negative (-; <10 fmol/mg of protein), positive (+; 10–200 fmol/mg of protein), or strongly positive (+ +; >200 fmol/mg of protein). nr, number.

 
Overexpression of Myb Protein.
Amplification of MYB was accompanied not only by overexpression of its mRNA but its protein product as well. Immunohistochemical staining of a xenograft from tumor 13996 (Fig. 1, A and B)Citation indicated strong nuclear immunostaining. A corresponding xenograft from breast cancer cell line T47D (non-BRCA1 mutation-positive) without MYBamplification showed no nuclear immunostaining (data not shown).

cDNA Microarrays.
The microarrays consisted of 4688 cDNA clones selected from the UniGene collection. The clones included 2629 homologous to known genes and 2059 clones with no known homology. Analysis of the BRCA1 tumor 13996 (with a high level of MYB amplification, 11.2-fold; Table 1Citation ) showed MYB (v-myb avian myeloblastosis viral oncogene homologue) expression to be greatly increased as well (15-fold; Table 2Citation ), relative to the MYB expression in the reference MCF-10A cells. Furthermore, several putative MYB- or cell cycle-regulated genes [i.e., cdc2, cyclin B1, aurora, and retinoblastoma-like 1 (p107) protein] were determined to be overexpressed as well (Table 2)Citation . Other putative candidate genes localized to the 6q22-q24 region, including MAP/ERK kinase 5, interferon-{gamma} receptor {alpha}, phosphodiesterase I/nucleotide pyrophosphatase 1, gap junction protein {alpha}1, myristylated alanine-rich protein kinase C substrate (MARCKS, 80K-L), connective tissue growth factor, and several anonymous ESTs, were not found to be overexpressed. However, a gene designated pleomorphic adenoma gene-like 1 and mapped to 6q24-q25, was found to be 8-fold overexpressed in tumor 13996. A complete list of clones and hybridization results from tumor 13996 is available on request, and can also be found at the National Human Genome Research Institute Web site.6


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  		Table 2 cDNA microarray analysis of BRCA1 tumor 13996 with high-level MYB gene amplification

Shown are a subset of the genes found to have high expression in the tumor 13996 relative to the reference MCF-10A cells. In the lower part of the Table are genes selected based on their known importance in cell cycle control, DNA repair, or suggested as being regulated by MYB. A complete list of clones and hybridization results is available on request.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Results from the present study suggest the MYB oncogene as a possible target gene in the 6q22-q24 amplicon found by CGH analysis of a breast tumor from a BRCA1 germ-line mutation carrier and subsequently by FISH analysis in additional BRCA1tumors (5 of 17; 29%). In contrast, MYB amplification was not found in 8 BRCA2 tumors or in 13 breast cancer cell lines, and in only 2 of 100 sporadic breast tumors. One earlier study reports a similar low incidence (1 of 59) of MYBamplification in sporadic breast cancer (11) . Moreover, infrequent observations of MYB amplification have been made in acute myelogenous leukemia, and colon and pancreas carcinoma (10 , 12 , 13) .

The MYB gene was found to be overexpressed in amplified tumors using Northern blot and cDNA microarray analysis, and c-Myb protein overexpression was detected by immunohistochemistry. The amplicon did not extend to the 6q25 region as evidenced by a normal copy number of the ER{alpha} gene in these tumors. Other 6q23 candidate genes and ESTs present in the array were not found to be overexpressed. We cannot rule out the importance of other target genes in the 6q23 region, including IGFBP4, a DNA-binding protein; A20, an elongation factor homologue; and additional ESTs not present on the microarray. However, the fact that MYB appeared as one of the most highly overexpressed genes among the ~5000 clones analyzed supports the assumption that 6q23 amplification in BRCA1 tumors is driven by this oncogene.

The Myb proteins are ancient regulators of gene expression, consisting of three discrete functional domains responsible for sequence-specific DNA binding, transcriptional activation, and negative regulation of the protein, and they function as key regulators of cell growth and differentiation (14) . The MYB oncogene was first discovered in avian retroviruses that cause acute leukemia; it encodes v-Myb proteins that are truncated and deregulated versions of the cellular c-Myb protein. The normal c-Myb protein is highly expressed in immature hematopoietic cells, and its expression decreases dramatically during cell differentiation (15) . B-Myb, one of two closely related MYB genes present in vertebrates, is a ubiquitously expressed key regulator of cell cycle progression, and ectopic expression of B-Myb protein was shown to override p53-induced G1 arrest, allowing cell cycle progression under circumstances when DNA repair or apoptosis normally would prevail (16) . Like B-Myb, A-Myb is also regulated by cyclin-dependent phosphorylation during the G1-S and S-phases, but (similar to c-Myb) it exhibits a more restricted tissue pattern of expression (14) . It was recently shown that mice homozygous for an A-MYB germ-line mutation develop to term but show defects in growth of certain tissues (17) . Male A-MYB- -/- - mice were infertile because of a block in spermatogenesis and the pachytene phase of meiosis, whereas females manifested poor mammary gland morphogenesis, mainly during pregnancy, and progesterone-induced ductal branching and alveolar outgrowth (17) . A similar lack of pregnancy-induced breast epithelial cell proliferation, duct branching, and alveoli development was observed in mice with conditional BRCA1 knockout in mammary tissue (18) . Thus, both BRCA and MYB proteins are crucially involved in the processes of DNA recombination and mammary gland development (2 , 17 , 18) .

The single MYB homologue present in Drosophila (Dm myb) and in Schizosaccharomyces pombe (cdc5) has been implicated as having a role in G2 progression and G2-M transition (19 , 20) . It was found that wing cells in Dm myb mutants enter and complete DNA synthesis but are blocked in G2 or at G2-M transition where they continue to replicate their DNA, resulting in polyploidy. This suggests that Dm myb possesses two cell cycle checkpoint functions, one in regulation of the G2-M transition and a second in prevention of endoreplication and maintenance of diploidy (19) . In all likelihood, Dm myb represents the progenitor of all three MYB genes evolved in vertebrates and encompasses their combined functions. Thus, in addition to tissue-specific expression, the different vertebrate MYB genes may possess both distinct and overlapping functions in cell cycle G1-S progression and G2-M control, as well as in cell differentiation. A potential role of BRCA1 in a G2-M checkpoint has also emerged (2) , and its seems reasonable to suspect an interplay between MYB and BRCA protein functions.

Homozygous disruption of the MYB gene in mice is lethal and causes death from severe anemia at day 15 of embryogenesis, which demonstrates the critical role of c-Myb during hematopoiesis and erythroid cell differentiation (21) . However, c-Myb expression has also been noted in nonhematopoietic cells, including normal and tumorigenic human breast epithelial cells and breast tumors (22 , 23) . Surprisingly, c-Myb expression in breast cells was found to be associated with estrogen stimulation and the presence of ER, possibly because of a posttranscriptional stabilization of the MYB transcript. Results from the present study support a relationship between c-Myb expression and ER-positive phenotype. ER-positive sporadic and BRCA2 tumors without MYB amplification displayed moderate c-Myb expression in Northern blot analysis, whereas ER-negative sporadic tumors exhibited low or no c-Myb expression. Thus, c-Myb may, like A-Myb, play a role in hormone-regulated growth and differentiation of breast epithelial cells and ER-positive breast cancer. However, because BRCA1 tumors usually are ER-negative (24) , the activity of MYB (single or amplified copy number) in BRCA1-deficient cells would be regulated by other pathways. Possibly, MYB amplification in BRCA1 tumors may reflect a compensatory mechanism to execute vital cellular functions or to override a cell cycle block caused by DNA damage. Providing further suggestive evidence for a link between MYB and BRCA1 regulation, several potential MYB binding sites (PyAACG/TG) are found in the BRCA1 promotor region.7

cDNA microarray analysis of BRCA1 tumor 13996 with high-level MYB amplification disclosed high expression of a multitude of cell cycle regulators, some of which have been suggested previously as being activated by MYB, including cdc2/cdc28, topoisomerase II {alpha}, cyclin B1, p53, MSH2, mitotic feedback control protein MAD2-like 1, aurora/IPL-1-related kinase, and retinoblastoma protein homologue p107 (14) . This deregulated gene expression may be a consequence of other cellular processes and should not be taken as evidence for a MYB-related activity. For example, tumor 13996 carries a somatic p53 mutation (Ser215Ile; data not shown). However, it provides a possible link between a transcription factor and its target genes and demonstrates the usefulness of microarray analysis in depicting cellular signaling pathways.

In conclusion, amplification of the MYB gene at 6q22-q24 was unexpectedly found to be a prevalent genetic aberration in BRCA1-associated breast tumors. This is an infrequent finding in sporadic breast cancer, and indeed, both sporadic and hereditary breast cancer usually are characterized by deletions on chromosome 6q (1 , 25) . Other oncogenes such as the ERBB2 oncogene, commonly found amplified in (ER-negative) breast cancer, are not associated with BRCA1 tumor development (24) . This illustrates that gene amplification is not merely a result of genomic instability and suggests a functional role of the c-Myb transcription factor in BRCA1 breast tumor progression. However, MYB is not essential for progression of BRCA1-related breast tumors because the majority of tumors examined lacked MYB amplification or overexpression. Moreover, we cannot rule out the existence of another target gene in the 6q22-q24 amplicon.


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

1 The present study was supported by grants from the Swedish Cancer Society, the Nordic Cancer Union, Mrs. Berta Kamprads Foundation, the Gunnar Arvid & Elisabeth Nilsson Foundation, the Crafoord Foundation, the Hospital of Lund Foundations, the F & M Bergqvist Foundation, the King Gustav V:s Jubilee Foundation, the Finnish Cultural Foundation, the Maud Kuistila Foundation, the Sigrid Juselius Foundation, and the Finnish Cancer Society. Back

2 Contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Department of Oncology, University Hospital, SE-22185 Lund, Sweden. Phone: 46 46 177569; Fax: 46 46 147327; E-mail: ake.borg{at}onk.lu.se Back

4 The abbreviations used are: CGH, comparative genomic hybridization; ER, estrogen receptor; FISH, fluorescence in situ hybridization; PAC, P1 artificial chromosome; PgR, progesterone receptor; EST, expressed sequence tag. Back

5 Johannsson et al., manuscript in preparation. Back

6 http://wwwdev.nhgri.nih.gov/MYB/. Back

7 http://www2.ncbi.nlm.nih.gov/genbank. Back

Received 2/17/00. Accepted 8/17/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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