Results

RT-PCR revealed PTTG1 expression in both hormone-related and unrelated cancer models. A specific 316-bp product for PTTG1 was demonstrated in all the human tumor cell lines tested (Figure 1). In breast cancer cell lines (6) expression for PTTG1 -mRNA was not related to highly invasive ability (BT-549), tumorogenicity (T47D, MDA^68), or positive ERa status (MCF-7, T47D, PM1).

expression in human tumor cell 298lines. Molecular weight markers VI

Lane 5, K562; Lane 6, Jurkat; Lane * 7, DLD1; Lane 8, LS513; Lane 9,

Lanel 1, BT549; Lane 12, MCF7.

DNA sequencing of PCR products obtained from cell lines JJPF-OJC4 and JCA-OJC3 were performed. A BLAST search confirmed a 98 and 99 % identity between the input sequence from JJPF-OJC4 and JCA-OJC3, respectively, and the data base sequence for human PTTG-1. Specificity (Table 3) of the PTTG1-mRNA assay was examined by PCR amplification of cDNA obtained from normal lymph nodes, BM, and peripheral blood (cellular and plasmatic mRNA).The PTTG1 transcript was not detected in control lymph nodes (n = 34), using 500 and 800 ng of LN-derived cDNA for PCR amplification.

Table 3. Specificity of the PTTGl-mRNA RT-PCR Assay.

Table 3. Specificity of the PTTGl-mRNA RT-PCR Assay.

A semi-quantitative approach to evaluate the expression for PTTG-1 in BM was used. Total RNA was derived from two lots of pooled normal human blood marrow (male/female; Lot 1, n = 83 and Lot 2, n = 36). PCR amplification was performed using Ampliwax and "hot start" PCR. Different amounts of input RNA from Lot 1 were used for reverse transcription and a constant fraction (25%) of the cDNA was amplified subsequently in the PCR. Expression for PTTG1 was demonstrated with input RNA ranged from 600 to 2000 nanograms.

Figure 2. Specificity ofPTTGl mRNA analysis. Low level of PTTG 1 mRNA expression (Lane 4) in blood from two healthy controls (A and B). Lane 1, (32-microglobulin; Lanes 2 and 3, negative results for epidermal growth factor receptor mRNA and cytokeratin 20 mRNA, respectively. MW Marker VIII (Roche).

PTTGl-mRNA was detected in peripheral blood in 4/14 healthy donors analyzed. Mean mRNA concentration was similar (165 ng ± 111.8, sd) in both positive and negative samples (p = NS, parametric and nonparametric test) (Figure

2). PCR and PTTG 1-specific primers amplified different amounts of cDNA obtained after reverse transcription of pooled BM-derived RNA of lot 2. No PTTG1 expression was detected with cDNA ranged from 600 to 2000 ng (Figure steqalive Controls Bone Marrow cDNA Colon Celi-Line JJPF-OJC4

Figure 3. PCR and PTTGl-specific primers amplified different amounts ofcDNA of pooled BM-derived RNA (n = 36). Lanes 1-5: Negative controls. Lanes 6 and 11: ß2-microglobulin (cDNA = 600 ng). Lanes 7-10: No PTTG1 expression was detected in BM, cDNA ranged from 600 ng. (Lane 7, 800 ng; Lane 8, 1 pg; Lane 910, 2 pg). Lanes 12-15: PTTG1 expression in colon cancer cell-line JJPF-OJC4 with cDNA. (Lane 12, 600 ng; Lane 13, 800 ng; Lane 14, lpg; Lane 15, 2 pg MW Marker VI/VIII (Roche).

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