Expression of the matrix metalloproteinase 9 in Hodgkin's disease is independent of EBV status

Abstract

Background: In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix.

Aim: To test whether the expression of MMP-9 in Hodgkin's disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin's disease cell line, L428.

Methods: MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin's disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkin's disease cell line, L428.

Results: MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkin's disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkin's disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin's disease cell line. Induction of LMP-1 expression in the Hodgkin's disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography.

Conclusions: These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkin's disease tumours and by the Hodgkin's disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.

Figure 1

Immunohistochemistry for matrix metalloproteinase 9 (MMP-9). Paraffin wax embedded section of Hodgkin's disease showing strong positive staining in the malignant Hodgkin and Reed-Sternberg cells.

Figure 2

(A) Western blot analysis demonstrates that latent membrane protein 1 (LMP-1) expression can be induced in L428 cells transfected with the LMP-1 gene (F1L428 cells) by incubation with 6 μM cadmium chloride for time periods ranging from zero hours (lane 1) to 24 hours (lane 6). Protein was detected using the CS1-4 monoclonal antibody reagent. (B) Western blot analysis of the L428 Hodgkin's disease cell line using the monoclonal reagent CS1-4. Lane 1 represents protein from the parental L428 Hodgkin's disease cell line, lane 2 is from L428 cells transfected with vector only, and lane 3 the vector plus LMP-1 insert (F1L428 cells). LMP-1 expression was induced in the F1L428 cells by incubation with 6 μM cadmium chloride for six hours and shows a protein band corresponding to LMP-1. As expected, treatment of parental L428 cells and H2 cells with cadmium produced no such band. (C) Zymography analysis of matrix metalloproteinase 9 (MMP-9) expression. Conditioned media from: lane 1, control cells showing bands for active and inactive forms of MMP-9; lane 2, L428 cells with LMP-1 insert (F1L428) uninduced; lane 3, F1L428 cells after induction of LMP-1 expression; lane 4, H2L428 cells (vector only); lane 5, L591 cells; lane 6, KMH2 cells.

Figure 3

Kaplan-Meier plots showing the survival curves for the three groups of Hodgkin's disease with differing intensities of matrix metalloproteinase 9 (MMP-9) expression (+, ++, and +++). There were no significant differences between the groups.