Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Apr;117(4):1019-28.
doi: 10.1172/JCI30945. Epub 2007 Mar 15.

Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas

Grzegorz Sarek  1 , Sari Kurki, Juulia Enbäck, Guergana Iotzova, Juergen Haas, Pirjo Laakkonen, Marikki Laiho, Päivi M Ojala
Affiliations

Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas

Grzegorz Sarek et al. J Clin Invest. 2007 Apr.

Abstract

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent for primary effusion lymphoma (PEL), a non-Hodgkin type lymphoma manifesting as an effusion malignancy in the affected individual. Although KSHV has been recognized as a tumor virus for over a decade, the pathways for its tumorigenic conversion are incompletely understood, which has greatly hampered the development of efficient therapies for KSHV-induced malignancies like PEL and Kaposi's sarcoma. There are no current therapies effective against the aggressive, KSHV-induced PEL. Here we demonstrate that activation of the p53 pathway using murine double minute 2 (MDM2) inhibitor Nutlin-3a conveyed specific and highly potent activation of PEL cell killing. Our results demonstrated that the KSHV latency-associated nuclear antigen (LANA) bound to both p53 and MDM2 and that the MDM2 inhibitor Nutlin-3a disrupted the p53-MDM2-LANA complex and selectively induced massive apoptosis in PEL cells. Together with our results indicating that KSHV-infection activated DNA damage signaling, these findings contribute to the specificity of the cytotoxic effects of Nutlin-3a in KSHV-infected cells. Moreover, we showed that Nutlin-3a had striking antitumor activity in vivo in a mouse xenograft model. Our results therefore present new options for exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment modality for this virally induced lymphoma.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Nutlin-3a activates p53 and its target genes in PEL cells.
KSHV-infected PEL cells (BC-1, BC-3, and BCBL-1), EBV-transformed LCLs (CZE and IHE), and cells with mutant p53 (DG-75 and HL-60) were incubated for 12 hours in the presence (+) or absence (–) of 7 μM Nutlin-3a. Whole-cell lysates were subjected to SDS-PAGE followed by Western blotting and analyzed for p53, MDM2, phosphorylated-p53(Ser15) [p-p53(Ser15)], 53BP1, p21CIP1, and Bax expression. Actin immunoblot is shown as a loading control.
Figure 2
Figure 2. Nutlin-3a induces cell-cycle arrest in PEL cells and EBV-transformed LCLs.
Asynchronously growing PEL cells (BC-1, BC-3, and BCBL-1; A), EBV-transformed LCLs (CZE and IHE; B), and mutant p53 cells (DG-75 and HL-60; C) were treated with 7 μM Nutlin-3a or vehicle control for the indicated time periods. Cells were pulse-labeled with BrdU and analyzed for DNA content by flow cytometry. BrdU incorporation during the S phase is indicated as percentage of stained cells. The sub-G1 populations in PEL cells are denoted by arrows. Data are representative of 3 independent experiments.
Figure 3
Figure 3. Nutlin-3a has cytotoxic activity in PEL cells.
(A) PEL cell lines (BC-1, BC-3, and BCBL-1) and KSHV-infected LCL IHH (green symbols), EBV-transformed LCLs (CZE and IHE; blue symbols), or mutant p53 cells (DG-75 and HL-60; red symbols) were cultured for 5 days with 7 μM Nutlin-3a. Cell viability was determined by trypan blue exclusion at the indicated time points. Results are shown as survival curves denoting percentage of viable cells relative to the vehicle control. Data represent the mean of 3 independent experiments. (B) Scatter plot of annexin V–FITC/PI flow cytometry of BC-1 cells after exposure to 7 μM of Nutlin-3a or vehicle control for different time periods. (C) Apoptosis in BC-3, BCBL-1, CZE, IHE, DG-75, and HL-60 cells was assessed at 96 hours after treatment with 7 μM Nutlin-3a or vehicle control by annexin V–FITC/PI binding and measured by flow cytometry analysis. Lower left quadrants represent viable cells (annexin V– and PI-negative); lower right quadrants represent early apoptotic cells (annexin V–positive, PI-negative) demonstrating cytoplasmic membrane integrity; upper right quadrants represent nonviable, late apoptotic cells (annexin V– and PI-positive). Numbers indicate the percentage of cells in each quadrant. Shown is 1 representative experiment of 3.
Figure 4
Figure 4. Nutlin-3a selectively kills KSHV-infected cells.
(A) U2OS and EA.hy 926 cells in the absence or presence of latent rKSHV infection were treated with 7 μM Nutlin-3a, and cell death was assessed by trypan blue exclusion at 24, 48, and 96 hours of treatment. Values represent the percentage of dead cells induced by Nutlin-3a treatment. The percentage of dead cells in the DMSO control was subtracted as a background. Each value represents the mean of 3 independent experiments. (B) Noninfected and rKSHV-infected EA.hy 926 cells were incubated for 12 hours in the presence or absence of 7 μM Nutlin-3a. Whole-cell lysates were subjected to SDS-PAGE followed by Western blotting and analyzed for p53 and MDM2 expression. Actin immunoblot is shown as a loading control.
Figure 5
Figure 5. DNA damage signaling enhances the cytotoxic effect of Nutlin 3a.
(A) BC-3 and CZE cells were immunostained with antibody against γH2AX, the phosphorylated form of histone H2AX. The nuclear morphology was visualized by Hoechst staining. (B) Percentage of γH2AX-positive cells in PEL cells (BC-1, BC-3, and BCBL-1) and EBV-transformed LCLs (CZE and IHE). Results represent the mean of 2 independent experiments. (C) BC-1, BC-3, BCBL-1, CZE, IHE, DG-75, and HL-60 cells were incubated for 12 hours in the presence or absence of 7 μM Nutlin-3a. Whole-cell lysates were subjected to SDS-PAGE followed by Western blotting and analyzed for phosphorylated Chk2(Thr68) and total Chk2 expression. (D) Survival curves for BC-1, IHH, and IHE cells (some gamma-irradiated at 1 Gy; IR) incubated with Nutlin-3a in the presence or absence of caffeine (2 mM). Cell death was determined by trypan blue exclusion at 24, 48, and 72 hours after treatment. Values represent the percentage of viable cells relative to that of DMSO control.
Figure 6
Figure 6. Nutlin-3a disrupts the p53-MDM2-LANA interaction.
(A) Extracts from the BC-3 cell line exposed for 12 hours to vehicle control or 7 μM Nutlin-3a were separated using gel filtration chromatography. Fractions were analyzed by Western blotting with antibodies against p53, MDM2, and LANA. The elution profile of molecular weight standards is indicated above the lanes. (B) The peak fractions for LANA (asterisks in A) from BC-3 cells treated with either vehicle control or 7 μM Nutlin-3a for 12 hours were used in immunoprecipitations with anti-p53 or anti-MDM2 antibodies. As a control, a duplicate sample from the same fraction was immunoprecipitated with mouse IgG. Immunocomplexes were resolved by SDS-PAGE and analyzed by Western blotting with antibodies against p53, MDM2, and LANA.
Figure 7
Figure 7. Antitumor activity of Nutlin-3a in vivo.
Growth curves of Nutlin-3a– and vehicle control–treated BC-3 tumors. Balb/c nude mice were injected subcutaneously with 6 × 106 BC-3 cells. When the tumors had grown to palpable size, the mice were treated intraperitoneally with the vehicle control (filled squares) or 20 mg/kg of Nutlin-3a (open circles). Nutlin-3a treatment resulted in regression of tumors; at the end of treatment, these tumors were significantly smaller than those treated with vehicle (P = 0.02). Dashed line indicates the volume of Matrigel in the tumor. Data (mean and SEM) are representative of 2 independent experiments. Inset shows a photograph of BC-3 tumor–bearing Balb/c nude mice treated with either vehicle control or 20 mg/kg Nutlin-3a 7 times over the course of 2 weeks.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chang Y., et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science. 1994;266:1865–1869. - PubMed
    1. Soulier J., et al. Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman’s disease. Blood. 1995;86:1276–1280. - PubMed
    1. Cesarman E., et al. In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi’s sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood. 1995;86:2708–2714. - PubMed
    1. Ablashi D.V., Chatlynne L.G., Whitman J.E., Jr., Cesarman E. Spectrum of Kaposi’s sarcoma-associated herpesvirus, or human herpesvirus 8, diseases. Clin. Microbiol. Rev. 2002;15:439–464. - PMC - PubMed
    1. Cesarman E., Knowles D.M. The role of Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) in lymphoproliferative diseases. Semin. Cancer Biol. 1999;9:165–174. - PubMed

Publication types

MeSH terms