Reed-Sternberg cells in Hodgkin's lymphoma present features of cellular senescence

Abstract

Hodgkin's Lymphoma (HL) is one of the most prevailing malignancies in young adults. Reed-Sternberg (RS) cells in HL have distinctive large cell morphology, are characteristic of the disease and their presence is essential for diagnosis. Enlarged cells are one of the hallmarks of senescence, but whether RS cells are senescent has not been previously investigated. Here we show that RS cells have characteristics of senescent cells; RS cells in HL biopsies specifically express the senescence markers and cell cycle inhibitors p21^Cip1 and p16^INK4a and are negative for the proliferation marker Ki-67, suggesting that these cells have ceased to proliferate. Moreover, the RS-like cells in HL lines, stained specifically for senescence-associated β-galactosidase (SA-β-gal). Oxidative stress promoted senescence in these cells as demonstrated by their staining for p21^Cip1, p16^INK4a, p53 and γH2AX. Senescent cells produce copious amounts of inflammatory cytokines termed 'senescence-associated secretory phenotype' (SASP), primarily regulated by Nuclear Factor κB (NF-κB). Indeed, we show that NF-κB activity and NF-κB-dependent cytokines production (e.g., IL-6, TNF-α, GM-CSF) were elevated in RS-like cells. Furthermore, NF-κB inhibitors, JSH-23 and curcumin reduced IL-6 secretion from RS-like cells. Thus, defining RS cells as senescent offers new insights on the origin of the proinflammatory microenvironment in HL.

Figure 1

Expression of senescence markers in biopsies of classic Hodgkin's lymphomas. Patients' biopsies show expression of the cell cycle inhibitors p21^Cip1 and p16^INK4a and NF-κB phospho-p65 in RS cells. RS cells are not stained by the cell proliferation marker Ki-67 (arrows)

Figure 2

The number of large RS cells is increased by oxidative stress. Cells were treated with 50 μM H₂O₂ for 2 h followed by 96 h incubation in growth medium. Cells of different size were determined by flow cytometry measurement of the cell forward-angle light scatter (FSC-A). To discriminate between large and small cells, the cutoff value equal to 120 AU (arbitrary units) was determined to correspond to the mean number of morphologically detected large cells in control cell cultures. (a) The results of a representative experiment (out of four independent reproducible experiments) depict the percentage values of large cells in control (3.3%) and in H₂O₂-treated cultures (28.7%) of L428 cells. (b) H₂O₂- treated cells were cytospined and stained by H&E. Arrows: H, Hodgkin small cells, mRS, mononuclear large RS and bRS, binuclear large RS cells

Figure 3

The senescence marker β-gal is exhibited by large RS cells. (a) Oxidative stress increased the number of large RS cell stained by β-Gal. Cells were treated with 50 μM H₂O₂ for 2 h followed by 96 h incubation in growth medium and adhered on slides by cytospin centrifugation, and β-gal was detected as described in Materials and Methods. (b), Large RS cells express increased levels of β-gal. Control and H₂O₂-treated L428 cells were stained by the β-gal substrate C₁₂FDG and the fluorescence of unsorted (all cells) and sorted small (H) and large (RS like) cells was analyzed by flow cytometry. A representative experiment is depicted. The bar graph represents the mean fluorescence intensity (MFI, %) and ±S.D. of three independent experiments. Significance of **P<0.05 was determined by t-test. (c), Large RS-like cells have limited proliferation capacity. The florescent dye CFDA-SE is retained in non-dividing cells and is diluted out in proliferating cells. Cells were labeled by CFDA-SE and fluorescence was detected after 96 h as described in Materials and Methods. The cells were photographed using Olympus confocal microscope 96 h after labeling ( × 600)

Figure 4

The senescence markers p16^INK4a and p21^Cip1 are expressed in RS-like cells. Cells were treated with 50 μM H₂O₂ for 2 h followed by 96-h incubation in growth medium. The cells were adhered on slides by cytospin centrifugation and stained by immunofluorescence with specific antibodies to p16INK4a (a) and p21Cip1 (b) as described in Materials and Methods. Cells were photographed using Olympus confocal microscope ( × 600)

Figure 5

RS-like cells exhibit high NF-κB activity. (a) NF-κB luciferase activity was determined in non-sorted L428 cells and in FACS-sorted H and RS cells in the presence or absence of H₂O₂ as described in Materials and Methods. (b) RS like cells are the main source for cytokine secretion. Control and H₂O₂ treated cells were FACS sorted as in (a). Supernatants were collected 24 h after plating followed by ELISA. Large RS cells expressed higher levels of IL-6, GM-CSF and TNF-α compared with H cells (calculated for each individual cytokine). (c) NF-κB is a modulator of IL-6 production in RS cells. Control and H₂O₂-treated cells were FACS sorted as in a. The specific NF-κB inhibitor JSH-23 (100 μM), Curcumin (30 μM) and Bortezomib (10 nM), were added to sorted cells and supernatants were collected after 24 h. IL-6 was determined in triplicates by ELISA and normalized to 10⁶ cells. The experiments were repeated three times and mean and ±S.D. were calculated. Significance of at least **P<0.05 was determined by t-test