Chang et al. 10.1073/pnas.0704366104. |
Fig. 5.
Generation of MMTV-Ptn-IRES-GFP transgenic mice. (A) Schematic diagram of the construct used for microinjection. MMTV, mouse mammary tumor virus promoter; hPtn, human pleiotrophin open reading frame; IRES, internal ribosomal entry site; EGFP, enhanced green fluorescence protein; SV40 PolyA, SV40 polyadenylation signal. (B) RT-PCR analysis of Ptn mRNA in mammary glands of different founder lines: 1, wild-type male; 2, wild-type female; 3, founder line 1 male; 4, founder line 1 female; 5, founder line 2 male; and 6, founder line 2 female.
Fig. 6. Fold changes of tropoelastin and protocollagen mRNA in MMTV-PyMT-Ptn transgenic mouse breast cancers compared with MMTV-PyMT mouse breast cancers. The primers used were mouse tropoelastin: forward, 5¢-GCC TGG GAA AGT TCC TGG TG-3¢, and reverse, 5¢-CCT TGG CTT TGA CTC CTG TG-3¢; mouse protocollagen Ia1: forward, 5'-GCA AGA ATG GAG ATG ATG GGG-3', and reverse, 5'-AAA CCA CTG AAG CCT CGG TGT C-3'; mouse protocollagen Ia2: forward, 5'-TGT AAA CAC CCC AGC GAA GAA C-3', and reverse, 5'-GAG TTG CCA TTT CCT TGG AGG -3'; mouse protocollagen IVa5: forward, 5'-CAG AGC ATC CAG CCA TTC ATT AG -3', and reverse, 5'-TAG CCA ATC CAC AGA GAG TCC CAC-3'; mouse protocollagen XIa1: forward, 5'-TGT ATG ATG GCT GTG CGT CTC G-3', and reverse, 5'-CCG ACT TCA AAT CCA AAC TTC TGG-3'; and mouse GAPDH: forward, 5¢-CCT GCA CCA CCA ACT GCT TA-3¢, and reverse, 5¢-TCA TGA GCC CTT CCA CAA TG-3¢ .
Fig. 7. PTN expression and secretion-dependent epithelial island formation. (A) Western blot of whole cell lysates of MCF-7 cells transfected with siRNAs against Ptn or control (scrambled) siRNAs probed with anti-PTN and with anti-actin antibodies. (B) Expression of PTN in whole cell lysates (WL) of MCF-7-PTN-KDAS cells (lane 1), expression of PTN in whole cell lysates (WL) of MCF-7-PTN-KDEL cells (lane 2), expression of PTN in conditioned media (CM) of MCF-7-PTN-KDAS cells (lane 3), and expression of PTN in conditioned media (CM) of MCF-7-PTN-KDEL cells (lane 4).
Table 1. Percentage of the areas occupied by scirrhous and nonscirrhous subtypes of invasive ductal carcinomas in breast cancers from MMTV-PyMT and MMTV-PyMT-Ptn mice (H&E)
Mice | Scirrhous area, % | Nonscirrhous area, % |
MMTV-PyMT (N = 22) | 22.5 ± 9.1 | 19.2 ± 15.8 |
MMTV-PyMT-Ptn (N = 25) | 38.9 ± 5.0* | 16.8 ± 16.6 |
The remaining areas in these breast cancers consist of areas of less advanced breast cancers, including ductal hyperplasia, adenoma/mammary intraepithelial neoplasia, and early carcinomas, as described by Lin et al. (1). *, P < 0.05.
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1. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW (2003) Am J Pathol 163:2113-2126.
Table 2. IMD and vessel size of breast cancers from MMTV-PyMT and MMTV-PyMT-Ptn mice
Mice | IMD, counts/mm2 | Microvessel size, mm2 |
MMTV-PyMT (N = 22) | 18.83 ± 10.2 | 393 ± 109 |
MMTV-PyMT-Ptn (N = 25) | 27.14 ± 13.5 | 658 ± 272* |
*, P < 0.01.
SI Materials and Methods
Transfections and Constructs.
MCF-7-Ptn cells, MCF-7-mock cells, MCF-7-Ptn-KDEL cells, and MCF-7-Ptn-KDAS cells were obtained by introducing the expression vectors pCDNA3.1-Ptn, pCDNA3.1 "empty vector," pCDNA3.1-PTN KDEL, or pCDNA3.1-PTN KDAS into MCF-7 cells. The C-terminal KDEL is an endoplasmic reticulum "retention sequence" (PTN-KDEL) that prevents secretion of PTN (1). The C-terminal KDAS is not a retention sequence but serves as a "scrambled" control to KDEL. The oligonucleotide sequences for KDEL [AGT(S) GAA(E) AAG(K) GAC(D) GAG(E) CTG(L) TAA] and for KDAS (control) [AGT(S) GAA(E) AAG(K) GAC(D) GCC(A) TCG(S) TAA] were added at the 3¢ terminus of Ptn.
The sequences of RNAi for the human Ptn gene are sense, 5-GCA GGG AAG AAA GAG AAA CUU-3; antisense, 5-GUU UCU CUU UCU UCC CUG CUU-3; scrambled control sequence: sense, 5-GCA GAG AGG AGA AAG AAA CUU-3; antisense, 5-GUU UCU UUC UCC UCU CUG CUU-3.
Transgenic Constructs, Transgenic Mice.
The Ptn cDNA was inserted into the pIRES-EGFP vector (BD Clontech, Mountain View, CA). The DNA was injected into the fertilized eggs of FVB mice. FVB/N-Tg(MMTV-PyMT)634Mul/J transgenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and have been described previously (2). Mice were examined daily after 4 weeks of age. Tumor length (L) and width (W) were measured, and tumor volume was estimate using the formula (L ´ W2)/2. The tumors that formed in their mammary glands were excised in 13-week-old animals.
Male athymic nude mice (8 weeks old, Cby.Cg-Foxn1nu; The Jackson Laboratory) were used. MCF-7-Mock or MCF-7-Ptn cells (2 ´ 106) were injected s.c. into the flanks of nude mice with or without 2 ´ 106 NIH 3T3 cells. The sites of injection were measured daily beginning 1 day after injection.
Western Blot Analysis.
Cells were rinsed in PBS, lysed in T-PER tissue protein extraction (Pierce, Rockford, IL) or Nonidet P-40 cell lysis buffer [10 mM Hepes (pH 7.2), 142.5 mM KCl, 1 mM EGTA, 5 mM MgCl2, and 0.5% Nonidet P-40] supplemented with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with appropriate antibodies. Anti-PTN antibodies were purchased from R&D Systems (Minneapolis, MN). HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Gelatin Zymography.
To obtain conditioned media, 200,000 cells were used. Protein concentration in supernatants was determined with the BCA protein assay kit (Pierce). Proteins from conditioned media were separated using SDS-PAGE containing 1 mg/ml gelatin. The gels were washed for 1 h in a buffer containing 50 mM Tris×HCl (pH 7.4) with 2% Triton X-100 to remove the SDS and incubated for 24 h at 37°C in the reaction buffer [50 mM Tris×HCl (pH 7.4), 10 mM CaCl2, 1% Triton X-100, and 0.02% NaN3] and stained for 30 min with 0.1% Coomassie brilliant blue R250 (ICN Biomedicals) to visualize bands of proteolytic activity.
In Situ
Hybridization.The human Ptn cDNA sequence was subcloned into the pCRII vector (Invitrogen, Carlsbad, CA). The plasmids were linearized. The sense and antisense probes were synthesized with a digoxigenin (DIG)-RNA Transcription Kit (Roche Diagnostics). Deparaffinized sections were incubated with 20 mg/ml proteinase K in 10 mM Tris/EDTA and treated with 0.2 M HCl for 10 min. After dehydration, sections were incubated with a hybridization solution in a moist chamber, washed in 2´SSC (standard saline citrate; 1´ SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7) containing 50% formamide, 2´SSC, and 0.2´SSC, incubated with a 1.5% blocking reagent and then with alkaline phosphatase-conjugated anti-DIG antibodies (Boehringer Mannheim, Mannheim, Germany). The sections were washed twice in Tris-buffered saline and developed. Negative controls of in situ hybridization included pretreatment of the sections with 100 mg/ml RNase A or with no probe and hybridization of the sections with the sense probe.
IMD.
Sections of breast cancers stained with anti-CD31 antibodies were used to measure microvessels per microscope field at ´400 magnification. The results were analyzed using test and control groups. The size of microvessels was measured using SPOT RT 3.4 software.
1. Munro S, Pelham HR (1987) Cell 48:899-907.
2. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW (2003) Am J Pathol 163:2113-2126.