Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expressing immortalized B cells - PubMed (original) (raw)

Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expressing immortalized B cells

H Höfelmayr et al. J Virol. 2001 Mar.

Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-Jkappa/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells.

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Figures

FIG. 1

Tetracycline-regulated mNotch1-IC expression in EREB2-5 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of EREB2-5 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (EREB-Notch cl.1 and cl.4) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of estrogen and tetracycline. (B) EREB2-5 cells and cells from EREB-Notch clones 1 and 4 were transiently transfected with a promoter-reporter construct containing a multimerized RBP-Jκ binding site. As negative control, a promoter-reporter gene construct without an RBP-Jκ binding site was used. Cells were grown in the presence of estrogen and presence or absence of tetracycline. Four hours before transfection, cells were washed to withdraw estrogen. After transfection, the cells were cultivated for 2 days in the presence (+E) or absence (−E) of estrogen to regulate the function of EBNA2 and in the presence (+N) or absence (−N) of tetracycline to regulate Notch1-IC expression. Luciferase activities were determined as relative light units (RLUs). Mean values and standard deviations of two independent experiments are indicated.

FIG. 2

mNotch1-IC stably expressed in EREB2-5 cells cannot maintain B-cell proliferation. After estrogen withdrawal, the same number of viable EREB and EREB-Notch clone 1 and clone 4 cells in the presence or absence of tetracycline (N), leading to the absence (−N) or presence (+N) of Notch1-IC were seeded in 96-well plates, and the number of viable cells was determined by staining with MTT reagent on the days indicated after estrogen withdrawal. As a positive control, the OD values of the parental line EREB in the presence of estrogen (+E) are shown. At day 5, fresh medium was added to all cells. To compare different MTT assays, OD values at day 1 were normalized to 1. Mean values and standard deviations are shown for triplicates.

FIG. 3

mNotch1-IC stably expressed in EREB2-5 cells cannot induce LMP1 expression. Protein extracts were prepared from EREB-Notch clones 1 and 4 in the presence and absence of estrogen and tetracycline. The protein extracts were Western blotted and analyzed by immunostaining with monoclonal anti-LMP1 antibody S12.

FIG. 4

Tetracycline-regulated mNotch1-IC expression in 1194 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of 1194 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (1194-Notch clones 1 and 2) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of tetracycline. The antibody detected a protein with the expected molecular mass of about 70 kDa, whereas an unspecific signal was visible in all lanes at about 80 kDa. (B) Extracts from the controls EREB-pHEBo and 1194-pHEBo and from the 1194-Notch clone 1 in the presence and absence of estrogen and tetracycline were analyzed by Western blots using monoclonal anti-LMP1 antibody S12.

FIG. 5

In the absence of estrogen, 1194-Notch cells continue to proliferate for 4 days. (A) 1194-Notch clone 1 cells were kept in the absence of estrogen for up to 7 days, and the number of viable cells was determined by MTT conversion on the days indicated. The rate of MTT conversion at day 0 was set to 1, and OD values were normalized accordingly. (B) After estrogen withdrawal, the same number of viable 1194-Notch clone 1 cells in the presence or absence of estrogen (E) and tetracycline (N), leading to the presence or absence of EBNA2 (+E and −E, respectively) and Notch1-IC (+N and −N, respectively), were seeded in 10-ml cell culture flasks. Cell growth was determined by counting the number of living cells on days 0, 2, 4, and 7 in triplicates. Dead cells were excluded by trypan blue dye staining. (C) The viability of the cells was determined as the ratio of the number of living to total cells. Average values of four experiments are shown. The standard deviations never exceeded 24% of the respective single values.

FIG. 6

1194-Notch cells pass through the cell cycle for 6 days. 1194-Notch (A) and 1194-pHEBo (B) cells were stained with propidium iodide 0, 3, 4, 6, 9, or 13 days after estrogen withdrawal. The relative numbers of cells in the G1 and G2 phases of the cell cycle were determined by FACS analysis.

FIG. 7

mNotch1-IC cannot upregulate c-Myc expression in 1194 cells. Extracts from 1194-pHEBo and 1194-Notch cells (clone 1) grown in the presence and absence of tetracycline were prepared 0, 1, and 2 days after estrogen withdrawal. The protein extracts were Western blotted and analyzed by immunostaining with the anti-Myc monoclonal antibody 9E10.

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Activated Notch1 can transiently substitute for EBNA2 in the maintenance of proliferation of LMP1-expressing immortalized B cells - PubMed (original) (raw)