Epstein-Barr virus (EBV) genome and expression in breast cancer tissue: effect of EBV infection of breast cancer cells on resistance to paclitaxel (Taxol)

Abstract

The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.

FIG. 1.

Laser capture microdissection (LCM) of breast cancer tissue. Frozen tumor sections (10 μm thick) stained with Mayer's hematoxylin and eosin Y were microdissected with the Arcturus PixCell II system to procure homogeneous cell populations. Panel A shows a section before LCM. Panel B shows clusters of the malignant epithelial cells captured by LCM. These are representative sections from the total microdissections summarized in Table 3.

FIG. 2.

Detection of EBV transcripts by RT-PCR analysis in RNA preparations from breast cancer biopsies. The primer combinations used are shown as arrows in the diagrams presented in panel A. For cDNA detection, the following primers were used: E1AS and E1S were used for EBNA-1 (a), LMP1S and LMP1AS for LMP-1 (b), and BARF1S BARF1AS for BARF-1 (c), and ZS and ZAS1 for BZLF1 (e). For detection of putative DNA contamination, BamAS and BamAAS (c) and U172AS and UPUS (d) were used as controls. Products obtained after RT-PCR are shown in panel B after Southern blotting (Ba, Bb, and Bd) or on agarose gels stained with ethidium bromide (Bc). Amplification of EBNA-1 cDNA is shown in panel Ba. MDA-MB-231 cells were used as the negative control, and EBV-infected MDA-MB-231 cells were used as the positive controls for EBNA-1 cDNA amplification. LMP-1 cDNA amplification is shown in panel Bb. B95-8 and C3B4 (an EBV-infected MDA-MB-231 cell line) served as positive controls. BARF-1 cDNA amplification is shown in panel Bc, and BZLF1 amplification is shown in panel Bd. B95-8 cDNA served as a positive control for BARF-1 and BZLF1, and DNA from B85-8 served as a positive control for DNA amplification in BamH1A and BamH1U.

FIG. 3.

Comparative toxicity of paclitaxel to MDA-MB-231 cells and to the different EBV-infected clones. Thirty thousand cells were seeded in 100 μl of culture medium in microtiter plates and incubated for 72 h with various concentrations of paclitaxel. The viability was assessed by means of the endogenous enzyme hexosaminidase. The experiment was performed in triplicate.

FIG. 4.

EBV infection up-regulates MDR1 expression in two different EBV-infected MDA-MB-231 cell clones. Quantitative real-time RT-PCR analysis was performed on RNA extracted from cells exposed during 72 h to 0.1 μM paclitaxel; the transcript copy numbers relative to the reference gene β2-microglobulin (b2m) were determined.