Transforming potential of the herpesvirus oncoprotein MEQ: morphological transformation, serum-independent growth, and inhibition of apoptosis

Abstract

Marek's disease virus (MDV) induces the rapid development of overwhelming T-cell lymphomas in chickens. One of its candidate oncogenes, meq (MDV Eco Q) which encodes a bZIP protein, has been biochemically characterized as a transcription factor. Interestingly, MEQ proteins are expressed not only in the nucleoplasm but also in the coiled bodies and the nucleolus. Its novel subcellular localization suggests that MEQ may be involved in other functions beyond its transcriptional potential. In this report we show that MEQ proteins are expressed ubiquitously and abundantly in MDV tumor cell lines. Overexpression of MEQ results in transformation of a rodent fibroblast cell line, Rat-2. The criteria of transformation are based on morphological transfiguration, anchorage-independent growth, and serum-independent growth. Furthermore, MEQ is able to distend the transforming capacity of MEQ-transformed Rat-2 cells through inhibition of apoptosis. Specifically, MEQ can efficiently protect Rat-2 cells from cell death induced by multiple modes including tumor necrosis factor alpha, C2-ceramide, UV irradiation, and serum deprivation. Its antiapoptotic function requires new protein synthesis, as treatment with a protein synthesis inhibitor, cycloheximide, partially reversed MEQ's antiapoptotic effect. Coincidentally, transcriptional induction of bcl-2 and suppression of bax are also observed in MEQ-transformed Rat-2 cells. Taken together, our results suggest that MEQ antagonizes apoptosis through regulation of its downstream target genes involved in apoptotic and/or antiapoptotic pathways.

FIG. 1

Constitutive expression of MEQ proteins in the MDV tumor cell lines and MEQ-transformed Rat-2 cells. Total cell lysates were prepared from MDV cell lines (RP1, RP4, RP19, and MSB1), MDV-infected T-cell lines (CU14 and CU41), reticuloendotheliosis virus cell lines (CU91, CU205, and RP13), avian leukosis virus cell line (RP9), and normal avian embryo fibroblasts (CEF and DEF) (A) as well as from MEQ-transformed and vector-infected Rat-2 cells (B). Cell lysates equivalent to approximately 0.5 × 10⁶ to 1 × 10⁶ cells per lane were analyzed by SDS–10% PAGE. The gels were transferred to Immobilon polyvinylidene difluoride membranes and blotted with MEQ antisera (1:4,000 dilution). The Western blots were subsequently detected with the conventional alkaline phosphatase method.

FIG. 2

Transformation of Rat-2 cells by MEQ. Rat-2 cells were infected with supernatants containing viruses derived from either pBabe-puro vector or pBabe-MEQ construct. The transforming potential of MEQ was evaluated by morphology (A and B) and soft agar colony assay (C and D). Magnification, ×55.

FIG. 3

MEQ-induced serum-independent growth in MEQ-transformed Rat-2 cells. A total of 2 × 10⁵ MEQ-transformed and the same number of vector-infected Rat-2 cells were cultured in the absence of serum for up to 4 days. The cell number was counted at 24 h intervals, in duplicate.

FIG. 4

MEQ-mediated BrdU incorporation and inhibition of apoptosis in serum-starved MEQ-transformed Rat-2 cells. BrdU was added to the media of MEQ-transformed and vector-infected Rat-2 cells for 12 h after cells were serum starved for 3 days. Cells were then fixed and stained with anti-BrdU MAb (A′ and B′) and counterstained with DAPI (A and B). Meanwhile, a TdT assay was used to evaluate apoptosis. Briefly, MEQ-transformed and vector-infected Rat-2 cells were also serum starved for 3 days, fixed and stained with ApopTag (C′ and D′) and counterstained with DAPI (C and D). Magnification, ×216.

FIG. 5

Protection of Rat-2 cells from apoptosis by MEQ. MEQ-transformed and vector-infected Rat-2 cells were treated with a variety of apoptosis-inducing vehicles including serum withdrawal, TNF-α, C2-ceramide (C2), UV irradiation, and CHX. Apoptosis was assessed with DAPI staining. Magnification, ×344.

FIG. 6

Statistical analysis of MEQ-mediated inhibition of apoptosis. The percent apoptotic cells was calculated based on the results shown in Fig. 5. C2, C2-ceramide.

FIG. 7

Induction of bcl-2 and suppression of bax expressions in MEQ-transformed Rat-2 cells. (A) Total cell lysates were prepared from MEQ-transformed Rat-2 cells grown in the presence of serum (lane 1) or after treatment with C2-ceramide in the absence of serum (lane 2) and vector-infected Rat-2 cells (in the presence of serum) (lane 3) and analyzed by SDS-PAGE. The Western blots were stained with rabbit anti-Bcl-2 and -Bax antisera and then detected with a Tropix Western-Light chemiluminescent kit. (B) RT-PCR was performed on total RNA extracted from MEQ-transformed Rat-2 cells grown in the presence of serum (lane 1) or after treatment with C2-ceramide in the absence of serum (lane 2). RT-PCR was similarly carried out with vector-infected Rat-2 cells grown in the presence of serum (lane 3). GAPDH gene expression was used as an internal control for the quality and quantity of the RT-PCR products.