Functional p53 signaling in Kaposi's sarcoma-associated herpesvirus lymphomas: implications for therapy

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.

FIG. 1.

PEL lines are sensitive to doxorubicin. (A to G) PEL-derived cell lines were seeded in triplicate at 2 × 10⁵ cells/ml. The indicated dosage of doxorubicin (in micrograms/milliliter) and/or vehicle (double-distilled H₂O) was added to each sample. Twenty-four hours postseeding and treatment, a sample was removed and cell survival was scored by MTT assay. Similar MTT measurements were performed every 24 h as indicated. Raw MTT numbers were normalized to the average reading of the untreated sample at day 1. Data shown represent three independent points, with error bars indicating the standard deviations. WT, wild type.

FIG. 2.

Doxorubicin induces cell cycle arrest in PEL lines. (A to F) PEL lines were seeded at 2 × 10⁵ cells per ml and treated the following day with 0.025 μg/ml doxorubicin. Cells were harvested 0, 8, 24, and 48 h posttreatment. The cells were fixed, and DNA content was analyzed by propidium iodide (PI) flow cytometry. Shown are histograms representing the cell cycle profile of ≥10,000 cells. WT, wild type.

FIG. 3.

Doxorubicin treatment of PEL lines activates the p53-mediated DNA damage response. (A) PEL lines were seeded at 2 × 10⁵ cells per ml in a total of 2 volumes of 40 ml each. The following morning, the cells were treated with 0.025 μg/ml doxorubicin and an initial aliquot of 20 ml was immediately obtained (t = 0 h). At the indicated time points following drug addition, cells were collected and pellets were frozen. Lysates from the time course were quantified, and equal amounts of protein were loaded for SDS-polyacrylamide gel electrophoresis analysis. Gels were transferred and blotted for the indicated protein products. Here, CDK6 is utilized as a loading control, p21 as a marker for p53 transcriptional activity, and phosphoserine 15 as an indicator of upstream DNA damage signaling. (B) Using densitometry, p21 expression was normalized to CDK6 (loading control), and the relative induction over levels observed at 0 h was determined for each time point.

FIG. 4.

PEL response to doxorubicin is p53 dependent. (A) BC-3 cells were seeded in triplicate at 2 × 10⁵ cells per ml in a 12-well dish with the indicated amount of pifithrin-alpha (PIF) or vehicle (DMSO). Following 24 h of pretreatment, 0.025 μg/ml doxorubicin (DOX) or vehicle (double-distilled H₂0) was added to the media. MTT assays were then performed every 24 h post-DOX addition as described in the legend to Fig. 1. Cell growth was normalized to the MTT reading of vehicle-treated cells on day 1. Points represent the average reading of three independent samples with error bars indicating the standard deviations. (B) Relative growth (percent vehicle control) at 72 h of culture for the indicated PEL cell lines either mock treated (black bars), PIF treated (gray bars), DOX treated (striped bars), or PIF and DOX treated (open bars). WT, wild type.

FIG. 5.

Nutlin-3 efficacy in PEL is Hdm2 and p53 dependent. (A to H) PEL lines were seeded at 2 × 10⁵ cells/ml in a total of 5 ml. The indicated dosage of Nutlin-3 (racemic) or vehicle (DMSO) was then added to the cells. Twenty-four, 48, 72, and 96 h post-Nutlin-3 addition, cell viability was determined using trypan blue exclusion. DG75 cells do not contain KSHV or EBV. (I) BCBL-1, BCP-1, and BC-3 cells were seeded in media containing 5 μM Nutlin or vehicle (DMSO) alone. Forty-eight hours later, samples were analyzed in quadruplicate for Annexin V positivity. Bar graphs represent the average Annexin V-positive population in each sample. Error bars show the standard deviation. WT, wild type. (J) Transcription profile analysis of Hdm2 transcript levels in PEL. Using Affymetrix data from 101 B-cell tumors and controls previously recorded by Klein et al. (40), we identified mRNAs that classify PEL away from all other tumors. These markers include the previously identified vitamin D receptor (K) and Hdm2 (L) and are shown using a bar graph representation of the average of median-centered mRNA levels for each tumor type. Each group represents ≥6 examples. (BL, Burkitt's lymphoma; DLBL, diffuse large B-cell lymphoma; CB, centroblastic; IB, immunoblastic). (M) PEL lines grown in complete media were harvested, and lysates were quantified. Equal protein quantities were separated by SDS-polyacrylamide gel electrophoresis, transferred, and immunoblotted for Hdm2, p53, and CDK6 (loading control) expression (top panel). Band intensities were quantified using ImageQuant software and normalized to CDK6. (N) The relative expression of Hdm2. (O) PEL lines were seeded, treated with 5 μM Nutlin-3, and harvested at increasing time points as described in the legend to Fig. 3. Following RNA isolation, cDNA was generated using random hexamer-primed reverse transcription. cDNA was subjected to quantitative real-time PCR for the indicated targets. Data were collected, and the relative change in gene expression was determined by calculating the difference between dCt1 and dCt2. Data were analyzed using ArrayMiner. Red represents gene induction relative to the median of all genes and data points in the array.

FIG. 5.

Nutlin-3 efficacy in PEL is Hdm2 and p53 dependent. (A to H) PEL lines were seeded at 2 × 10⁵ cells/ml in a total of 5 ml. The indicated dosage of Nutlin-3 (racemic) or vehicle (DMSO) was then added to the cells. Twenty-four, 48, 72, and 96 h post-Nutlin-3 addition, cell viability was determined using trypan blue exclusion. DG75 cells do not contain KSHV or EBV. (I) BCBL-1, BCP-1, and BC-3 cells were seeded in media containing 5 μM Nutlin or vehicle (DMSO) alone. Forty-eight hours later, samples were analyzed in quadruplicate for Annexin V positivity. Bar graphs represent the average Annexin V-positive population in each sample. Error bars show the standard deviation. WT, wild type. (J) Transcription profile analysis of Hdm2 transcript levels in PEL. Using Affymetrix data from 101 B-cell tumors and controls previously recorded by Klein et al. (40), we identified mRNAs that classify PEL away from all other tumors. These markers include the previously identified vitamin D receptor (K) and Hdm2 (L) and are shown using a bar graph representation of the average of median-centered mRNA levels for each tumor type. Each group represents ≥6 examples. (BL, Burkitt's lymphoma; DLBL, diffuse large B-cell lymphoma; CB, centroblastic; IB, immunoblastic). (M) PEL lines grown in complete media were harvested, and lysates were quantified. Equal protein quantities were separated by SDS-polyacrylamide gel electrophoresis, transferred, and immunoblotted for Hdm2, p53, and CDK6 (loading control) expression (top panel). Band intensities were quantified using ImageQuant software and normalized to CDK6. (N) The relative expression of Hdm2. (O) PEL lines were seeded, treated with 5 μM Nutlin-3, and harvested at increasing time points as described in the legend to Fig. 3. Following RNA isolation, cDNA was generated using random hexamer-primed reverse transcription. cDNA was subjected to quantitative real-time PCR for the indicated targets. Data were collected, and the relative change in gene expression was determined by calculating the difference between dCt1 and dCt2. Data were analyzed using ArrayMiner. Red represents gene induction relative to the median of all genes and data points in the array.