The pituitary tumor transforming gene 1 (PTTG-1): an immunological target for multiple myeloma

Abstract

Background: Multiple Myeloma is a cancer of B plasma cells, which produce non-specific antibodies and proliferate uncontrolled. Due to the potential relapse and non-specificity of current treatments, immunotherapy promises to be more specific and may induce long-term immunity in patients. The pituitary tumor transforming gene 1 (PTTG-1) has been shown to be a novel oncogene, expressed in the testis, thymus, colon, lung and placenta (undetectable in most other tissues). Furthermore, it is over expressed in many tumors such as the pituitary adenoma, breast, gastrointestinal cancers, leukemia, lymphoma, and lung cancer and it seems to be associated with tumorigenesis, angiogenesis and cancer progression. The purpose was to investigate the presence/rate of expression of PTTG-1 in multiple myeloma patients.

Methods: We analyzed the PTTG-1 expression at the transcriptional and the protein level, by PCR, immunocytochemical methods, Dot-blot and ELISA performed on patient's sera in 19 multiple myeloma patients, 6 different multiple myeloma cell lines and in normal human tissue.

Results: We did not find PTTG-1 presence in the normal human tissue panel, but PTTG-1 mRNA was detectable in 12 of the 19 patients, giving evidence of a 63% rate of expression (data confirmed by ELISA). Four of the 6 investigated cell lines (66.6%) were positive for PTTG-1. Investigations of protein expression gave evidence of 26.3% cytoplasmic expression and 16% surface expression in the plasma cells of multiple myeloma patients. Protein presence was also confirmed by Dot-blot in both cell lines and patients.

Conclusion: We established PTTG-1's presence at both the transcriptional and protein levels. These data suggest that PTTG-1 is aberrantly expressed in multiple myeloma plasma cells, is highly immunogenic and is a suitable target for immunotherapy of multiple myeloma.

Figure 1

1A. Scheme of pQE30 plasmid for the generation of PTTG-1 protein.1B. SDS-PAGE gel for purification of PTTG-1 protein stained with commassie blue. Line 1) washing buffer, 2) flow through fraction, 3) elution 2, 4) elution 1, 5) pQE30/pttg-1 with IPTG, 6) pQE30/pttg-1 without IPTG, 7) Marker.

Figure 2

2A. The PCR results of the normal tissue panel are shown. Except for the positive control (the testis), none of the analyzed organs showed positive band signals. 2B. IHC results did not show the presence of PTTG-1 at the protein level in the normal tissue array. Investigated tissues: 1) brain, 2) breast, 3) colon, 4) heart, 5) kidney, 6) liver, 7) lung, 8) ovary, 9) pancreas, 10) skeletal muscle, 11) spleen, 12) stomach and 13) bone marrow.

Figure 3

3A. i. The PCR result shows 12/19 positive cases for PTTG-1 at the transcriptional level.ii. Representative cases of patient MM plasma cells. In the (P) panel, the cytoplasmic staining is shown by ICC, IF and FACS. Also the NP panel shows a positive result for surface staining by ICC, IF and FACS methods. 3B. i. The investigation of the 6 established MM cell lines gave positive band signals in the four cases of KMS-11, 8226, ARK-B and ARP-1. ii. The evaluation of PTTG-1 protein expression showed only cytoplasmic but not surface positive reaction. The case of the permeabilized (P) and not permeabilized (NP) 8226 cell line is shown by means of ICC, IF and FACS.

Figure 4

A. Dot-blot confirms the PCR data in representative MM cell lines on the left (U266, 8226, ARP-1, ARK-B and KMS-11) and patients (negative patients not shown) on the right, in three different serial dilutions (1:100, 1:500, 1:1000). 1) positive ctrl (PTTG-1 20 μg), 2) positive ctrl (PTTG-1 10 μg), 3) negative ctrl (TBS only). The primary antibody was diluted 1:250 (Zymed Lab), secondary antibody 1:5000 (Pierce). B. ELISA technique shows the presence of IgG against PTTG-1 in 12/19 MM patients (63%). The cut-off point (mean + 3 STDEV), based on 11 healthy controls' values, is OD_{450 nm} = 0.404.