The pro-apoptotic protein Bax can homodimerize with itself and heterodimerize with the anti-apoptotic protein Bcl-2, but the significance of these protein-protein interactions remains unclear. Alanine substitution mutations were created in a well conserved IGDE motif found within the BH3 domain of Bax (residues 66-69) and the resulting mutant Bax proteins were tested for ability to homodimerize with themselves and to heterodimerize with Bcl-2. Correlations were made with cell death induction by these mutants of Bax both in mammalian cells where Bax may function through several mechanisms, and in yeast where Bax may exert its lethal actions through a more limited repertoire of mechanisms perhaps related to its ability to form ion channels in intracellular membranes. Two of the mutants, Bax(D68A) and Bax(E69A), retained the ability to homodimerize but failed to interact with Bcl-2 as determined by yeast two-hybrid assays and co-immunoprecipitation analysis using transfected mammalian cells. The Bax(E69A) protein exhibited a lethal phenotype in yeast, which could be specifically suppressed by co-expression of Bcl-2, despite its failure to dimerize with Bcl-2. Both the Bax(D68A) and Bax(E69A) proteins induced apoptosis when overexpressed in human 293 cells, despite an inability to bind to Bcl-2. Moreover, co-expression of Bcl-2 with Bax(D68A) and Bax(E69A) rescued mammalian cells from apoptosis. In contrast, a mutant of Bax lacking the IGDE motif, Bax(DeltaIGDE), was incapable of either homodimerizing with itself or heterodimerizing with Bcl-2 and was inactive at promoting cell death in either yeast or mammalian cells. Although failing to interact with Bcl-2, the Bax(D68A) and Bax(E69A) mutants retained the ability to bind to Bid, a putative Bax-activating member of the Bcl-2 family, and collaborated with Bid in inducing apoptosis. When taken together with previous observations, these findings indicate that (i) Bax can induce apoptosis in mammalian cells irrespective of heterodimerization with Bcl-2 and (ii) Bcl-2 can rescue both mammalian cells and yeast from the lethal effects of Bax without heterodimerizing with it. However, these results do not exclude the possibility that BH3-dependent homodimerization of Bax or interactions with Bax activators such as Bid may either assist or be required for the cell death-inducing mechanism of this protein.

Purpose

HIV-related diffuse large B-cell lymphoma (DLBCL) may be biologically different from DLBCL in the general population. We compared, by HIV status, the expression and prognostic significance of selected oncogenic markers in DLBCL diagnosed at Kaiser Permanente in California, between 1996 and 2007.

Experimental design

Eighty HIV-infected DLBCL patients were 1:1 matched to 80 HIV-uninfected DLBCL patients by age, gender, and race. Twenty-three markers in the following categories were examined using IHC: (i) cell-cycle regulators, (ii) B-cell activators, (iii) antiapoptotic proteins, and (iv) others, such as IgM. Tumor marker expression was compared across HIV infection status by Fisher exact test. For markers differentially expressed in HIV-related DLBCL, logistic regression was used to evaluate the association between tumor marker expression and 2-year overall mortality, adjusting for International Prognostic Index, cell-of-origin phenotype, and DLBCL morphologic variants.

Results

Expression of cMYC (% positive in HIV-related and -unrelated DLBCL: 64% vs. 32%), BCL6 (45% vs. 10%), PKC-β2 (61% vs. 4%), MUM1 (59% vs. 14%), and CD44 (87% vs. 56%) was significantly elevated in HIV-related DLBCLs, whereas expression of p27 (39% vs. 75%) was significantly reduced. Of these, cMYC expression was independently associated with increased 2-year mortality in HIV-infected patients [relative risk = 3.09 (0.90-10.55)] in multivariable logistic regression.

Conclusions

These results suggest that HIV-related DLBCL pathogenesis more frequently involves cMYC and BCL6 among other factors. In particular, cMYC-mediated pathogenesis may partly explain the more aggressive clinical course of DLBCL in HIV-infected patients.

Objective

Understanding tumor microenvironment and its impact on prognosis of HIV-related lymphomas may provide insight into novel therapeutic strategies.

Design

We characterized the relationship between infiltrating immune cells with tumor characteristics, HIV disease history and survival in 80 patients with HIV-related diffuse large B-cell lymphoma (DLBCL) diagnosed in the era of combined antiretroviral therapy (1996-2007) at Kaiser Permanente California. Eighty patients with HIV-unrelated DLBCL were included for comparison.

Methods

Data on patients' clinical history were obtained from Kaiser Permanente's electronic health records. The density of stromal CD4, CD8 and FOXP3 T cells and CD68 macrophages, as well as tumor molecular characteristics were examined using immunohistochemistry. The associations between stromal immune infiltration and patient's clinical history or tumor characteristics were examined using Kruskal-Wallis tests or Pearson's correlation coefficient. The effect of stromal immune infiltration on 2-year mortality was evaluated in multivariable logistic regression.

Results

Compared with HIV-unrelated DLBCL, patients with HIV-related DLBCL had significantly reduced stromal CD4 and FOXP3 T cells, but increased density of macrophages. Increased density of stromal macrophages was correlated with lower circulating CD4 cell count at DLBCL diagnosis. Tumor molecular characteristics, including BCL6, p53 and cMYC expression, but not Epstein-Barr virus infection status, were significantly correlated with stromal immune infiltration, particularly FOXP3 T cells. A higher density of infiltrating CD8 T cell was significantly associated with reduced mortality in patients with HIV-related DLBCL (odds ratio = 0.30 [0.09-0.97] for ≥25 vs. <10%).

Conclusion

These data provide evidence for the prognostic significance of cytotoxic T cells in determining outcomes of HIV-related lymphoma.

Purpose

Epstein-Barr virus (EBV)-mediated lymphomagenesis in the setting of HIV infection has been widely accepted. However, little is known about how EBV impacts prognosis. We investigated the hypothesis that EBV infection is associated with expression of specific B-cell oncogenic markers in HIV-related diffuse large B-cell lymphoma (DLBCL) and examined the prognostic use of detecting EBV infection.

Experimental design

HIV-related DLBCL cases diagnosed between 1996 and 2007 within Kaiser Permanente California were identified. Immunohistochemical staining was used to analyze the expression of selected markers that are cell-cycle regulators, B-cell activators, and antiapoptotic proteins among others. EBV infection was determined by in situ hybridization of EBV RNA. Correlations between EBV and marker expression were examined using Spearman correlation coefficient. The prognostic use of EBV status was examined in multivariable Cox model adjusting for International Prognostic Index (IPI). Receiver-operating characteristics (ROC) analysis was used to evaluate improvement in model discrimination.

Results

Seventy HIV-related DLBCL cases were included (31% EBV±). EBV+ tumor was associated with increased expression of BLIMP1 and CD30 and reduced expression of BCL6 and LMO2. EBV+ tumor was independently associated with elevated 2-year overall mortality [HR, 3.3; 95% confidence interval (CI), 1.6-6.6]. Area under the ROC curve showed improved model discrimination when incorporating tumor EBV status with IPI in the prediction model [0.65 vs. 0.74 (IPI only)].

Conclusion

Our results suggest that EBV infection was associated with expression of several tumor markers that are involved in the NF-κB pathway and that detecting tumor EBV status may have prognostic use in HIV-related DLBCLs.