Effects of rearrangement and allelic exclusion of JJAZ1/SUZ12 on cell proliferation and survival

Abstract

Polycomb group genes (PcGs) have been implicated in cancer based on altered levels of expression observed in certain tumors and the behavior of cultured cells containing inserted PcG transgenes. Endometrial stromal tumors provide evidence for a direct causal relationship because they contain several chromosomal translocations and resultant gene fusions involving PcGs, the most common of which joins portions of the JAZF1 gene to the PcGJJAZ1/SUZ12. We show here that both benign and malignant forms of this tumor have the JAZF1-JJAZ1 fusion but only the malignant form also exhibits exclusion of the unrearranged JJAZ1 allele. To evaluate the effects of both the JJAZ1/SUZ12 fusion and allelic exclusion on functions related to cell growth, we studied HEK293 cells that were modified with respect to JJAZ1 expression. We found that the JAZF1-JJAZ1 fusion restored levels of the polycomb protein EZH2 and histone 3 lysine 27 trimethylation, which were reduced by knockdown of endogenous JJAZ1. At the same time, the presence of JAZF1-JJAZ1 markedly inhibited apoptosis and induced above normal proliferation rates, although the latter effect occurred only when normal JJAZ1 was suppressed. Our findings suggest a genetic pathway for progression of a benign precursor to a sarcoma involving increased cell survival associated with acquisition of a PcG rearrangement, followed by accelerated cellular proliferation upon allelic exclusion of the unrearranged copy of that gene. Furthermore, these results indicate the likely functional importance of allelic exclusion of genes disrupted by chromosomal translocations, as seen in a variety of other cancers.

Fig. 1.

Analysis of JJAZ1 protein and mRNA in ESS cell lines. (A) Western blot of cell lines analyzed with anti-JJAZ1 antiserum. Cases 1, 2, and 3 are primary ESS cell lines. Chr, S2, and S3 represent the chromatin-enriched cell fraction, the soluble cytoplasmic fraction, and the soluble nuclear fraction, respectively. (B) Real-time RT-PCR for JJAZ1 mRNA. The amount of WT allele JJAZ1 mRNA was compared with the amount of β-actin mRNA. ESS1 is an ESS cell line derived from a tumor lacking the t(7;17).

Fig. 2.

Analysis of JAZF1 protein and mRNA in ESS cell lines. (A) Real-time RT-PCR for JAZF1 mRNA. The amount of WT JJAZ1 mRNA was normalized against the amount of β-actin mRNA. (B) Western blot of cell lines analyzed with anti-JAZF1 antiserum. Cases 1 and 2 are primary ESS cell lines.

Fig. 3.

Analysis of mRNA in SN cases. (A) RT-PCR for JAZF1–JJAZ1 fusion mRNA in SN cases 1–4 and a t(7;17)-positive tumor as a positive control. (B) Real-time RT-PCR for JJAZ1 mRNA in four SN cases as well as the t(7;17)-positive tumor case 4.

Fig. 4.

Effects of siRNA knockdown of JJAZ1 in 293 cells. (A) Western blot analyses of protein extracts from 293 cells stably infected with an pSiREN vector encoding siRNA targeted to JJAZ1 mRNA sequence 5′ to the fusion point (419-JJAZ1); a negative control siRNA that does not recognize any known human mRNA; an empty vector control of another siRNA vector system (pRetro-Super); and pRetro-Super containing an siRNA targeting JJAZ1 mRNA 3′ of the fusion point (pRetro-SuperSuz12). Histone 3 served as loading control. Compared with negative control or empty vector control, siRNA targets 5′ or 3′ of JJAZ1 substantially reduced JJAZ1, secondarily reduced EZH2 because of the presumed destabilization in the absence of JJAZ1, and modestly reduced trimethyl H3K27, as reported by others (21, 23). (B) Effects of JJAZ1 knockdown on 293 cell proliferation. Compared with negative control siRNA or empty vector, knocking down JJAZ1 slowed 293 cell growth. The y axis is calibrated in total cells, and the x axis is calibrated in days of growth. Cell numbers at each time point were determined by suspending cells in Hank's buffered saline after trypsinizing the cells growing in monolayers and counting suspended cells in a hemocytometer. The results shown were from experiments done in triplicate. Three additional experiments that were performed in triplicate yielded similar results.

Fig. 5.

Effects of JJAZ1 and JAZF1–JJAZ1 transgenes on 293 cells with and without knock down of endogenous JJAZl expression. (A) 293 cells that were infected previously with virus encoding siRNA targeting JJAZ1 mRNA 5′ of the fusion point (419-JJAZ1) were infected again with viral vectors expressing JJAZ1 (MIGRI-JJAZ1), JAZF1–JJAZ1 (MIGRI-J-J), or no insert (MIGRI-empty). Histone 3 served as loading control. Compared with empty vector control (MIGRI-empty), JAZF1–JJAZ1 reestablished EZH2 and H3K27 trimethylation to a level similar to that in parental 293 cells. (B) Effect of JAZF1–JJAZ1 on 293 cell proliferation after siRNA knockdown of JJAZ1 expression. Introducing JAZF1–JJAZ1 induced faster growth rate than parental 293 cell. (C) Effect of JAZF1–JJAZ1 on 293 cell proliferation without siRNA knockdown of JJAZ1 expression. JAZF1–JJAZ1 expression reduced the cell proliferation rate compared with empty vector control.

Fig. 6.

Cell survival after culture in serum-deprived media. (A) Total numbers of surviving cells per dish according to the protocol described under Materials and Methods, after initial seeding with 10⁴ cells. (B) Images of representative tissue culture dishes showing numbers of colonies after staining with crystal violet. Dishes were initially seeded with 2 × 10³ cells. The number adjacent to each dish indicates the total individual colonies counted in that dish under a low magnification. (C) Histogram of the numbers of colonies counted in experiments described for B.

Fig. 7.

Cell survival after culture under hypoxic conditions simulated by addition of desferrioxamine, as described under Materials and Methods. (A) Images of representative tissue culture dishes showing numbers of colonies after staining with crystal violet. Dishes were initially seeded with 2 × 10³ cells. Surviving colonies containing the JAZF1–JJAZ1 fusion tended to be confluent by the time any colonies in the control dishes appeared, thereby precluding accurate quantitative assessment of the survival advantage to cells with the JAZF1–JJAZ1 fusion, as in Fig. 6C. (B) FACS analysis of DNA content in cells stained with propidium iodide. The vertical arrows indicate the peak of sub2n cells, reflecting ongoing apoptosis in these cells. The horizontal axis represents the number of cells.