Proteomic analysis of mantle-cell lymphoma by protein microarray

Abstract

Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19(+) purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.

Figure 1.

Heat map with unsupervised clustering analysis of the 6 MCL samples compared with normal control. The MCL samples are numbered 1 to 6. The control sample is at the far right of the heat map. All the samples were normalized to the control sample using the Genespring software. The data points are colored by expression with lower signal values colored blue and higher values, red. Yellow signifies expression values equal to the control sample.

Figure 2.

Protein expression in the 6 MCL samples compared with normal control. The y-axis is a log scale of the normalized intensity. The data points are colored by expression with lower signal values colored blue and higher values, red and presented by their relative level in each sample compared with the normal control. The central line is the midpoint with the lines above and below at log-rank increase or decrease in expression. MCL7, which had a reversed expression, is presented on the right side of the control and is repeated twice. Note the reversed expression of the proteins in MCL7 as marked by the color changes. Data were analyzed with Genespring software.

Figure 3.

Expression levels of the 13 overexpressed proteins in the MCL samples compared with control. The data points are colored in red/orange indicating overexpression. The samples are numbered 1 to 7. MCL7 showed reversed expression pattern compared with the other 6 MCL samples. MCL7 was repeated twice and demonstrates consistent expression pattern. Data were analyzed with Genespring software.

Figure 4.

Immunoblotting analysis was performed to confirm the expression of several proteins identified in protein microarrays by using the rest of the total protein extracted for the microarray procedure. Protein from control B-lymphocyte extracts was loaded twice (normal tonsil) on each side of the gel. The nitrocellulose blots were probed with monoclonal anti-RCC1, p43/EMAPII precursor, procaspase-7, inhibitor 2, Hsp90, AKAP149, cyclin D1, Rb2, and procaspase-8. Loading control was performed with antiactin antibody (bottom row).

Figure 5.

Immunohistochemistry analysis to confirm the presence of the expression of the proteins in the MCL samples and confirm the absence of expression differences in other proteins not detected by the protein microarray. Paraffin-embedded tissue biopsies available from the same patients were analyzed using antibodies to human proteins: CRIK-L, Hsp90, and MDM2 (A); Rb2, KU80, Paxillin, and Bcl-x (B). Images were visualized under an Olympus IX70 inverted microscope equipped with objective lenses from 20 ×/0.40 and 60 ×/0.70 (Olympus, Tokyo, Japan). A MagnaFire digital camera (model 599806) and MagnaFire software version 4.1 were used to capture images (Olympus).

Figure 6.

Immunohistochemistry analysis of RCC-1, Hsp90, and MDM2 in several tonsillar tissues. This indicates that the expression of proteins detected in the control sample used in the array analysis is similar to other tonsillar tissues. Image acquisition was performed as in Figure 5.