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. 2002 May 28;99(11):7444-9.
doi: 10.1073/pnas.062189599.

A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo

Kimmo Porkka  1 , Pirjo Laakkonen, Jason A Hoffman, Michele Bernasconi, Erkki Ruoslahti
Affiliations

A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo

Kimmo Porkka et al. Proc Natl Acad Sci U S A. .

Abstract

We used a screening procedure to identify protein domains from phage-displayed cDNA libraries that bind both to bone marrow endothelial progenitor cells and tumor vasculature. Screening phage for binding of progenitor cell-enriched bone marrow cells in vitro, and for homing to HL-60 human leukemia cell xenograft tumors in vivo, yielded a cDNA fragment that encodes an N-terminal fragment of human high mobility group protein 2 (HMGN2, formerly HMG-17). Upon i.v. injection, phage displaying this HMGN2 fragment homed to HL-60 and MDA-MB-435 tumors. Testing of subfragments localized the full binding activity to a 31-aa peptide (F3) in the HMGN2 sequence. Fluorescein-labeled F3 peptide bound to and was internalized by HL-60 cells and human MDA-MB-435 breast cancer cells, appearing initially in the cytoplasm and then in the nuclei of these cells. Fluorescent F3 accumulated in HL-60 and MDA-MB-435 tumors after an i.v. injection, appearing in the nuclei of tumor endothelial cells and tumor cells. Thus, F3 can carry a payload (phage, fluorescein) to a tumor and into the cell nuclei in the tumor. This peptide may be suitable for targeting cytotoxic drugs and gene therapy vectors into tumors.

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Figures

Figure 1
Figure 1
Phage enrichment in vivo. A cDNA phage pool that was preselected for ex vivo binding to bone marrow cells was injected into the tail vein of mice. After 10 min of circulation, the mice were perfused through the heart, and phage was rescued from various organs, amplified, and used for subsequent rounds of selection. Fold enrichment of selected phage pool relative to the unselected cDNA phage library pool is shown.
Figure 2
Figure 2
Localization of the HMGN2-N cell binding site. The sequence and cell binding activity of individual HMGN2-N fragments. Inserts encoding for the various fragments were cloned into T7 phage, and the phage preparations were tested for binding to primary cells from HL-60 xenograft tumors. After a 1-h incubation at 4°C, the cells were washed and phage-quantified (the pfu are indicated above the columns). The enrichment is shown relative to a control phage (nonrecombinant T7 phage). One experiment representative of four is shown. The sequence encoded by exon 3 of HMGN2 is underlined.
Figure 3
Figure 3
Tissue localization of i.v.-injected F3 peptide. HMGN2-N F3 peptide and ARALPSQRSR control peptide were conjugated to fluorescein. Each labeled peptide was injected into the tail vein of mice bearing HL-60 or MDA-MB-435 xenografts. The peptide injection was followed 10 min later by an injection of biotinylated tomato lectin. After another 5 min, the mice were perfused through the heart with a fixative solution, and the organs were dissected, sectioned, and stained with streptavidin-Alexa 594. The slides were counterstained with 4′,6-diamidino-2-phenylindole and examined under an inverted fluorescent microscope. (e) An HL-60 tumor section from a mouse injected with fluorescein-labeled ARALPSQRSR control peptide, all other panels are from mice injected with fluorescein-labeled F3. (a) HL-60 tumor; (b) brain; (c) skin; (d) gut; (e) ARALPSQRSR control peptide in HL-60 tumor; (f) fluorescein-labeled F3 in an MDA-MB-435 tumor; (g) a higher magnification view from a showing the localization of F3 (green), lectin-stained vasculature (red), and 4′,6-diamidino-2-phenylindole-stained nuclei (blue). The green and blue images are shown individually in h and i. (Magnifications: a, b, and e, ×200; c and d, ×100; and f–i, ×400.)
Figure 4
Figure 4
FACS profile of bone marrow cells labeled with fluorescent F3 peptide and antibodies against cell differentiation markers. Fluorescein-labeled peptides, F3 and ARALPSQRSR control peptide at 2 μM, were incubated with gradient-depleted bone marrow cells and analyzed in a flow cytometer. (A) Control peptide (number/percentage of cells in lower right quadrant: 1/0.0); (B) F3 peptide (308/0.88); (C) F3 vs. CD45 (77% CD45-positive); (D) F3 vs. CD34 (75% CD34-negative).
Figure 5
Figure 5
Kinetics of uptake and nuclear translocation of F3 peptide in cultured MDA-MB-435 cells. The cells were incubated with fluorescein-labeled F3 (1 μM) for the indicated length of time at 37°C, washed, fixed, and examined under an inverted fluorescent microscope.
Figure 6
Figure 6
Nuclear localization of the L- and D-forms of the F3 peptide. Nuclear localization in HL-60 cells of F3 (a) or ARALPSQRSR control peptide (b). (c–f) Uptake by MDA-MB-435 cells of F3 synthesized either from L or D amino acids. The cells were treated as in Fig. 5A and stained with 4′,6-diamidino-2-phenylindole before examination under a confocal (a and b) or inverted fluorescent microscope (c–f). (c and e) Peptide staining (green); (d and f) nuclear staining (blue). (Magnifications: a and b, ×400; c–f, ×200.)
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