To assess the level of TVM expression (DR6 and Gpm6B) and explore its potential pro- and antiapoptotic role during tumour development, we examined the expression of DR6 and Gpm6B mRNAs by RT-PCR and Western blot analysis isolated from human peripheral whole blood.
The first TVM with antiapoptotic activity whose expression was examined is glycoprotein M6B (Gpm6B).
Amplification of the specific genes DR6, Gpm6B, and fi-actin ran for 30 cycles (94[degrees]C 5 min, 94[degrees]C 30 s, 58.4[degrees]C 30 s, and 72[degrees]C 45 s), using appropriate primers (Table 1) in the thermocycler 7500 RealTime PCR System (Applied Biosystems).
After western blotting (Trans blot SD semidry transfer cell, Biorad) blots on nitrocellulose membrane were probed with a rabbit monoclonal antibody against Gpm6B (Sigma, dilution 1:100) at room temperature for 12 hours and rabbit monoclonal antibody against DR6 (Sigma) at room temperature for 5 hours in dilution 1: 1000.
The results of molecular detection of DR6 and Gpm6B expression in patients with breast cancer were compared to control group with no tumour predisposition (n = 35).
In the next step of our work we analysed the mRNA expression of DR6, Gpm6B, and fi-actin genes in peripheral blood of patients (n = 75) with breast carcinoma (ductal invasive carcinoma n = 65, lobular invasive carcinoma n = 8, and nondifferentiated beast carcinoma n = 2).
During the detection of Gpm6B in the same group of patients against control group, we found significantly increased (18[+ or -]3.1%) levels of Gpm6B mRNA (Figure 1).
The last stage of our research was aimed at finding the correlation between grade of ductal invasive carcinoma (G1 to G3) and expression of DR6 and Gpm6B. In statistical analysis of DR6 mRNA level changes we detected significant increase starting in G1 (120 [+ or -] 3.2%) and reached maximal level in G3 (197 [+ or -] 6.5%) (Figure 3).
In the levels of Gpm6B mRNA from the blood of patients suffering from invasive lobular carcinoma (LIC) we determined increased levels against control group (40 [+ or -] 5.3%) (Figure 5).
The levels of protein Gpm6B were also significantly increased (22% [+ or -] 4.2%) against control group (Figure 6).
The variations in the expression of 17 genes (underlined in Table 2) are involved in specific neuronal pathways, such as neuronal cell structure (GPM6B
, PRPH, RELN), neural signaling and development (HOXB2, ELAVLI, EFNB1, NRG2, NELL1, MDK, GFRA1), synaptogenesis and steroid biosynthesis (SYCP2, PCDH17, STX1B2, SCG5, Nstage 4 diseasel, GATA3, NROB1).