Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-beta1 and IL-6

Abstract

Background: AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation.

Methods: Growth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3.

Results: Results confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1.

Conclusion: These studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3.

Figure 1

Southern blot analysis of the immunoglobulin heavy chain (IgH) locus in DNA from (A) LCL8664 cell line and (B) the original tumor mass from which LCL8664 cell line was derived. DNA (5 μg) was digested with Sacl and hybridized to a probe from the human IgH locus, 3' to the J_H region. The arrows indicate the fragments generated from the locus in germline organization. The asterisks indicate the clonal rearrangement of the IgH locus. DNA from the tumor mass contains a large germline fragment not present in the cell line, indicative of contamination of the original necropsy sample with non-neoplastic tissue.

Figure 2

(A). Dose-dependent growth inhibition of LCL8664 cells by TGF-β1. LCL8664 cells were cultured with TGF-β1 in varying concentrations from 0 ng/ml to 20 ng/ml. Viable cell counts were determined by trypan blue exclusion at regular intervals for 8 days. (B). Positive and negative growth regulation of LCL8664 cells by TGF-β1 and IL-6. LCL8664 cells were cultured with TGF-β1 (1 ng/ml), IL-6 (20 ng/ml), both, or were untreated (untr). Cell viability was quantified over 6 days of treatment using a tetrazolium dye reduction assay.

Figure 3

Activated (phosphorylated) STAT3 and ERK1/2 in LCL8664 cells treated with IL-6 and/or TGF-β1. LCL8664 cells were treated with IL-6 (20 ng/ml), TGF-β1 (1 ng/ml), both, or were untreated for time intervals between 0 – 60 minutes. Phosphorylated STAT3 (pSTAT3) and phosphorylated ERK1/2 (pERK1/2) were detected by intracellular flow cytometry.

Figure 4

Caspase-3 activity in LCL8664 cells treated with IL-6 and/or TGF-β1. LCL8664 cells were treated with IL-6 (20 ng/ml), TGF-β1 (1 ng/ml), both, or were untreated (untr) for 16 or 24 hours. Parallel samples were treated with the broad caspase inhibitor, zVAD-fmk (zVAD) as a negative control. Caspase-3 activity was quantified in whole cell lysates using a colorimetric assay. The data are reported as fold-change in caspase-3 activity relative to untreated samples.

Figure 5

(A).Bcl-2 expression in LCL8664 cells treated with IL-6. LCL8664 cells were treated with IL-6 (20 ng/ml), or were untreated (untr), for time intervals between 1 – 24 hours. Bcl-2 expression was examined by intracellular flow cytometry. As a positive control, Bcl-2 expression in Jurkat cells was detected using the same methodology. (B). Immunoblot analysis of Mcl-1 expression in LCL8664 cells treated with IL-6 and/or TGF-β1. In the left panel, LCL8664 cells were treated with IL-6 (20 ng/ml) for time intervals between 0 – 24 hours. In the right panel, LCL8664 cells were treated with IL-6 (20 ng/ml), TGF-β1 (1 ng/ml), both, or were untreated (C) for 4 hours. The expression of β-tubulin was examined as a loading control. (C). Activated (phosphorylated) Akt expression in LCL8664 cells treated with IL-6. LCL8664 cells were treated with IL-6 (20 ng/ml), or were untreated (untr), for time intervals between 0 – 60 minutes. Phosphorylated Akt (p-Akt) expression was examined by intracellular flow cytometry. As a positive control, p-Akt expression in Jurkat cells was detected using the same methodology, in the presence or absence of LY294002 (50 βM).

Figure 6

Positive and negative growth regulation of AIDS-NHL cell lines by TGF-β1 and IL-6. BCBL-1, UMCL01-101 and 2F7 cell lines were cultured with TGF-β1 (1 ng/ml), IL-6 (20 ng/ml), both, or were untreated. Cell viability was quantified at daily intervals thereafter using a tetrazolium dye reduction assay.

Figure 7

(A). Immunoblot analysis of LMP-1 and EBNA2 expression in 2F7 and UMCL01-101 (UMCL) cell lines. The expression of β-tubulin was examined as a loading control. (B). Bcl-6 expression in 2F7 and UMCL01-101 (UMCL) cell lines as examined by intracellular flow cytometry. The shaded and open histograms represent analysis with Bcl-6 antibody and isotype control antibody, respectively. (C). Activated (phosphorylated) STAT3 in 2F7 and UMCL01-101 cell lines. 2F7 cells were treated with IL-6 (20 ng/ml), or were untreated, for time intervals between 0 – 60 minutes. UMCL01-101 cells (UMCL) were treated with IL-6 (20 ng/ml), TGF-β1 (1 ng/ml), both, or were untreated, for time intervals between 0 – 60 minutes. Phosphorylated STAT3 (pSTAT3) was detected by intracellular flow cytometry.