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Materials and Methods:
Tissues and Cells
Histopathologically confirmed normal and breast cancer specimens were obtained as
fresh, frozen samples from the Pathology Department, Johns Hopkins Hospital and Duke
University (exemption granted by the respective Institutional Committees for use of
discarded patient material without identifiers). Freshly excised primary breast tumors were
trimmed of fat and muscle, minced fine with razor blades and digested with 0.15%
collagenase A (Boehringer Mannheim) and 0.5% dispase II (Boehringer Mannheim)
prepared in RPMI 1640 medium (Gibco/BRL). The cell clumps were separated from the
floating cells by allowing the cells to settle by gravity for 15 min in a 50 ml conical
centrifuge tube on ice, and then removing the supernatant (containing mainly fibroblasts) by
gentle suction. This process was repeated 2-3 times. The cell clumps were digested for 15’
with 0.25% trypsin, washed with RPMI 1640 containing 10% bovine serum, and
immunostained with anti-cytokeratin-specific antibody (CAM 5.2, Becton-Dickinson) to
assess the level of epithelial cell enrichment (Trask et al., 1990).
The epithelial cells
comprised between 70-80% of the enriched cell population.
Primary breast tumor samples were cryosectioned, and 5-10 micron sections were
screened by a pathologist after staining every tenth section with hematoxylin and eosin.
Sections containing more than 70% carcinoma cells were used for RNA and protein
extractions directly.
Normal epithelial cell strains and lines: The cell strains, 76N and H16N, were
derived from a normal individual’s mammary epithelium (Band et al., 1990 a) and were
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generous gifts from Dr. Vimla Band. Cultured normal finite lifespan breast epithelial cell
strains 184 and immortalized derivatives 184A1 and 184B5 were generous gifts of Dr.
Martha Stampfer (University of California, Berkeley, CA), were maintained according to
growth conditions described in (www.lbl.gov/mrgs ). HMEC finite lifespan strain 9F1403
was obtained from Clonetics.
Immortal HMECs, HBL100 and MCF10A were obtained
from ATCC (Rockville, MD).
Breast cancer cell lines: With the exception of EP, MW (Chu et al., 1985), MT, NT
and PT (Band et al., 1990 b), the breast cancer cell lines were obtained from American Type
Culture Collection (ATCC, Rockville, MD) and grown according to their recommendations.
Cell line MW1C6.3 is an agar subcloned, tumorigenic subline of MW breast cancer cells,
derived in our laboratory. Parental HCT116 colon carcinoma cells (p53+/+), 379.2, (a
derivative which has lost both alleles of p53 by homologous recombination), and 40.16 (a
derivative carrying a targeting vector integrated in another site in the genome) (Bunz et al,
1998) were grown in Mc Coy’s 5A medium (Gibco/BRL) supplemented with 10% fetal
bovine serum (Hyclone) and penicillin/streptomycin (Gibco/BRL). RKO, a colon cancer cell
line, and SAOS, an osteosarcoma cell line (ATCC) were grown in DMEM supplemented
with 10% fetal bovine serum (Hyclone).
Northern blot analysis
Five or 10 micrograms of total RNA, prepared from pulverized tissue specimens
using Trizol (Life Technologies, Inc.), was fractionated in a denaturing agarose gel,
transferred to a nylon membrane and probed with full length human p53 cDNA labeled with
32P]dCTP using the Rediprime random primer labeling kit (Amersham RPN 1633). A
300 bp DNA fragment specific for 18S rRNA or a 500 bp fragment of 36B4 cDNA, a human
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acidic ribosomal phosphoprotein gene (Marc E. Lippman, Lombardi Cancer Center) served
as a loading control. Densitometry was performed with the IPLabGel program (Signal
Analytics, Vienna VA) on a Macintosh microcomputer to obtain ratios of p53 and 18S rRNA
signals.
RNase Protection Assay
In vitro transcription of 32P] labeled riboprobes and RNase protection assays were
performed according to the manufacturer’s instructions (Ambion Inc.). The probes used
were transcripts of a 117 bp HOX cDNA fragment isolated by RT-PCR (Chariot and
Castronovo, 1995), and cloned into a TA (Invitrogen) vector. Transcripts of a 158 bp
fragment of 36B4 cDNA served as the RNA loading control. Following electrophoresis, the
gel was dried and exposed to film. Densitometry was performed with the IPLabGel program
(Signal Analytics, Vienna VA) on a Macintosh microcomputer to obtain ratios of HOXA5
and 36B4 signals.
Sequence Analysis
Promoter sequences obtained from GenBankTM were analyzed using the GCGTM software
program.
Human and mouse HOX Recombinant Plasmids
The four HOX cDNAs from pBSHOXA5 (John. F. Fuller, UCLA), pBSHOXB4
(Ching-Pin Chang, Stanford University), pBSHOXB5, and pBSHOXB7 (Corey Largman,
UCSF), were subcloned into the Kpn I and Xba I sites of the mammalian expression vector,
pCDM8 (Invitrogen). A Xho I-Sca I fragment that contains the entire mouse HoxA5 cDNA
(Tournier-Lasserve et al., 1989) was cloned into the Sma I site of pCDM8 (Invitrogen)
containing a modified polylinker, generating a plasmid designated pCMVHoxA5. Details of
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this construction are available on request. For the truncated protein, the pCMVHoxA5 was
digested with Bgl II, filled in with Klenow DNA polymerase, and religated. The 4 base
addition creates a stop codon at amino acid 187, eliminating sequences which include the
homeobox. This clone is referred to as pCMV∆HoxA5. The nature of the cloned fragments
were confirmed by nucleotide sequencing.
p53 Reporter Plasmids
The human p53/Luciferase constructs were generated by excising the 2.4 kb Xba I
fragment and the 356 bp Xba I-BamH I fragment from pICAT (Reisman et al., 1988),
followed by subcloning each fragment into the Sma I site of pGL2 basic Luciferase reporter
vector (Promega) The nature of the inserts in –356bp p53-Luc and –2.4kb p53-Luc were
confirmed by nucleotide sequencing.
For the construction of the mouse –320mp53CAT, a 676 bp Hind III-EcoR I
restriction fragment from a mouse genomic clone was subcloned into an engineered Sal I
cloning site in the vector pUC18CAT. To produce deletions in the promoter, a 2.4 kb Hind
III fragment from bacteriophage lambda DNA was inserted into the Hind III site located at
the 5' end of the p53 promoter in the polylinker of -320mp53CAT. The resulting plasmid (–
o
320 mp53CATSalH2.4) was linearized, and digested with Bal 31 nuclease at 30 C. The
DNAs were then digested with HindIII to release the remaining lambda DNA and the
plasmids were ligated in the presence of Sal I linkers so that the different promoter deletions
could be cloned into the pUC18CAT vector. The endpoints of the deletions were determined
by nucleotide sequencing.
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Transient transfection and luciferase and CAT assays
Human SAOS2 osteosarcoma cells, ZR75.1 breast cancer cells, or RKO colon
carcinoma cells, were seeded (5x105 cells) in 60 mm tissue culture dishes in DMEM
supplemented with 10% fetal bovine serum 24 h prior to transfection. The –2.4kb p53-Luc
or the –356bp p53-Luc constructs (2 µg) were transfected alone or with the pCMVHOXA5, B4, -B5 or -B7-expression plasmids (1 µg), using lipofection according to the manufacturer’s
instructions (PanVera).
Transfection efficiency for each assay was assessed by
cotransfection of 5 ng of Renilla Luciferase expression plasmid (Promega). Luciferase
activities were assayed 36 h after transfection using the Dual Luciferase Assay kit
(Promega). The p53 promoter-firefly luciferase generated light output was normalized to the
light output obtained with Renilla luciferase in each cell line.
SAOS2 human osteosarcoma cells, and RKO human colon carcinoma cells were
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plated at 5x10 /60mm dish 24 h prior to transfection and then transfected with 2 ugof –
320mp53CAT, –229mp53-CAT or –153mp53CAT, and/or 1ugexpression plasmid
pCMVHoxA5 or pCMV∆Hox as described above. Cell extracts were prepared 36 h after
transfection. CAT activity was assayed as described (Seed and Sheen, 1988).
Site Directed Mutagenesis
The core HoxA5 binding site in the -320mp53CAT construct was mutated using the
Altered Sites
TM
in vitro Mutagenesis System (Promega). Mutations in the promoter were
confirmed by nucleotide sequencing.
Gel-Shift Assays
Cell extracts from SAOS2 cells transfected with the expression vector pCMVHoxA5 were
prepared 24 h after transfection and gel-shift assays were performed as described (Raman et
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al., 1993) using 3 µg of cell extract. For the supershift assays, DNA/protein complex was
incubated with 2 µg of rabbit polyclonal HOXA5 antiserum, AB-2 (BABCO) for 10 min on
ice. Antigen/antibody complex was depleted using Protein A-G as described in the protocols
(Oncogene Sciences). Each probe was annealed to its complementary oligonucleotide before
EMSA.
Immunoblot Analysis
Proteins were visualized by Western analysis and 10% SDS-PAGE. The primary
antibodies [anti-HOXA5 (HOXA5-2, BABCO), anti-p53 (AB-7, Oncogene Science), or antiß-actin (AC-15, Sigma), rabbit polyclonal anti-p21 (15091A, Pharmingen), anti-Mdm2
(65101A, Pharmingen), anti-PARP (AB-2, Oncogene Sciences), anti-dynein (Zymed) and
anti- Na+, K+ATPase (gift of Ed Benz, Johns Hopkins)] were used at 1:1000 dilution.
The inducible HOXA5 system
The two plasmid, ecdysone-inducible mammalian expression kit (Invitrogen), was
used to generate the clones. Briefly, the HOXA5 gene was cloned into the EcoRI site of the
vector, pIND, generating the plasmid, pINDHOXA5. MCF-7 cells were transfected with 2
ugeach of recombinant pINDHOXA5 and pVgRXR, or with 2 µg each of pIND and
pVgRXR using lipofectamine (PanVera). Six stable clones from each culture (designated
MCF7-HOXA5-1 to 6 and MCF7-VGX-1 to 6) were selected for G418 (Gibco/BRL) and
zeocin resistance. The inducibility of HOXA5 was assessed upon addition of Ponasterone A
(5 µM) by Western analysis using the polyclonal antibody, anti-HOXA5-2. Of the six
MCF7-HOXA5 clones, only two (MCF7-HOX-1, and -2) survived passage longer than two
months.
Analysis of cell morphology alteration, DNA fragmentation and PARP cleavage
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MCF7-HOX-1, and -2 were treated with Ponasterone A (5 µM) (Invitrogen) for 48 h.
MCF-7 cells were treated with the protease inhibitor, MG-132 (10 µM) (Peptide Institute,
Osaka, Japan) for 16 h. Cultures were photographed with a Zeiss Axioplan microscope.
DNA fragmentation analysis was performed on these cells as described (Li, 1997; Pandey,
1994). Immunoblot analysis for PARP cleavage using anti-PARP (AB-2, Oncogene
Sciences) was performed as described (Lazebnik et al, 1994, Li et al, 1997).
Colony forming assays
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5x10 HCT116 (p53+/+), 40-16 (p53+/+) and 379-2 (p53-/-) colon carcinoma cells
were plated in a 60 mm dish 24 h prior to transfection. Duplicate plates were transfected by
lipofection (as described above) with 1 µg of pCDM8, pCMVHOXA5 or pCMVp53 along
with 250 ng of pBOSH2BGFP (a plasmid that confers blasticidin resistance from Bert
Vogelstein). Twelve hours after transfection, each plate was split into three 100 mm plates
with medium containing 2 µg/ml of Blasticidin S (Calbiochem). Following selection for two
weeks, the colonies were stained with 0.1% trypan blue in methanol and counted.
Identical culture conditions were used to transfect plates of MCF-7 and ZR75.1 cells
with pCDM8, pCMVHOXA5 or pCMVHOXA5. Selection was carried out in G418containing medium, plates were stained with 0.1% trypan blue, and colonies were counted
after two weeks.
References for Methods:
Trask, D. K. et al. Keratins as markers that distinguish normal and tumor-derived mammary
epithelial cells. Proc Natl Acad Sci U S A 87, 2319-2323 (1990).
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Band, V., Zajchowski, D., Kulesa, V., and Sager, R.
Human papilloma virus DNAs
immortalize normal human mammary epithelial cells and reduce their growth factor
requirements. Proc Natl Acad Sci U S A 87, 463-467 (1990).
Chu, M. Y., et al.
Differential characteristics of two newly established human breast
carcinoma cell lines. Cancer Res. 45, 1357-1366 (1985).
Band, V. et al. Tumor progression in four mammary epithelial cell lines derived from the
same patient. Cancer Res 50, 7351-7357 (1990).
Bunz, F., et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage.
Science 282, 1497-1501 (1998).
Chariot, A., & Castronovo, V. Detection of HOXA1 expression in human breast cancer.
Biochem Biophys Res Commun. 222, 292-297 (1996).
Tournier-Lasserve, E., Odenwald, W. F., Garbern, J., Trojanowski, J., & Lazzarini, R. A.
Remarkable intron and exon sequence conservation in human and mouse homeobox Hox 1.3
genes. Mol Cell Biol 9, 2273-2278, (1989).
Reisman, D., Greenberg, M., & Rotter, V. Human p53 oncogene contains one promoter
upstream of exon 1 and a second, stronger promoter within intron 1. Proc Natl Acad Sci,
USA 85, 5146-5150 (1988).
Seed, B., and Sheen, J. Y.
A simple phase-extraction assay for chloramphenicol
acetyltransferase activity. Gene 67, 271-277 (1988).
Raman, V., Andrews, M. E., Harkey, M. A., and Raff, R. A. Protein-DNA interactions at
putative regulatory regions of two coordinately expressed genes, msp130 and PM27, during
skeletogenesis in sea urchin embryos. Int. J Dev. Biol. 37, 499-507 (1993).
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Li, F. et al. Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL
has activity independent of cytochrome c release. J. Biol. Chem. 272, 30299-30305 (1997).
Pandey, S., Walker, P.R. & Sikorska, M. Separate pools of endonuclease activity are
responsible for internucleosomal and high molecular mass DNA fragmentation during
apoptosis. Biochem Cell Biol. 72, 625-629 (1994).
Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., & Earnshaw, W. C.
Cleavage of poly (ADP-ribose)ploymerase by a proteinase with properties like ICE. Nature
371, 346-347 (1994).
Li, F. et al. Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL
has activity independent of cytochrome c release. J. Biol. Chem. 272, 30299-30305 (1997).
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