Elevated endogenous expression of the dominant negative basic helix-loop-helix protein ID1 correlates with significant centrosome abnormalities in human tumor cells

<p>Abstract</p> <p>Background</p> <p>ID proteins are dominant negative inhibitors of basic helix-loop-helix transcription factors that have multiple functions during development and cellular differentiation. Ectopic (over-)expression of ID1 extends the lifespan of primary human epithelial cells. High expression levels of ID1 have been detected in multiple human malignancies, and in some have been correlated with unfavorable clinical prognosis. ID1 protein is localized at the centrosomes and forced (over-)expression of ID1 results in errors during centrosome duplication.</p> <p>Results</p> <p>Here we analyzed the steady state expression levels of the four ID-proteins in 18 tumor cell lines and assessed the number of centrosome abnormalities. While expression of ID1, ID2, and ID3 was detected, we failed to detect protein expression of ID4. Expression of ID1 correlated with increased supernumerary centrosomes in most cell lines analyzed.</p> <p>Conclusions</p> <p>This is the first report that shows that not only ectopic expression in tissue culture but endogenous levels of ID1 modulate centrosome numbers. Thus, our findings support the hypothesis that ID1 interferes with centrosome homeostasis, most likely contributing to genomic instability and associated tumor aggressiveness.</p

A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches.

Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases

Endogenous protein levels of TIMPs in degenerative cervical NP cells.

<p>To determine the endogenous expression levels of TIMPs in cervical NP cells, 15 NP specimens of degenerative grade III and IV of herniated discs were used. In collagen I scaffold 4×10⁵ NP cells from each specimen were grown for four weeks, and on the basis of disc degeneration grade (DDG) the protein concentration of TIMPs were defined (ELISA) from 100 µg total protein extracts of each sample. TIMP-1 expression levels (Fig. 3a), TIMP-2 expression levels (Fig. 3b), TIMP-3 expression levels (Fig. 3c) and TIMP-4 expression levels (Fig. 3d) are shown by box plots with whiskers min to max.</p

Expression levels of anti-catabolic proteins TIMPs in degenerative NP cells.

<p>NP cells were isolated from 63 samples of degenerative IVDs and 4 x 10⁵ cells from each sample were cultured for four weeks in collagen I scaffold. Concentration data (ELISA) of TIMPs from 100 µg total protein extracts were statistically analyzed on the bases of disc degeneration grade (DDG). Box plots with whiskers min. to max. show TIMP-1 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4a</a>), TIMP-2 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4b</a>), TIMP-3 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4c</a>) and TIMP-4 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g004" target="_blank">Figure 4d</a>).</p

Levels of endogenous protein expression for catabolic and anti-catabolic cytokines in degenerative cervical NP cells.

<p>Levels of endogenous protein expression for catabolic and anti-catabolic cytokines in degenerative cervical NP cells.</p

Concentrations of endogenously expressed inflammatory cytokines, anabolic factors and matrix proteins in degenerative cervical NP cells.

<p>Concentrations of endogenously expressed inflammatory cytokines, anabolic factors and matrix proteins in degenerative cervical NP cells.</p

Expression levels of catabolic proteins MMPs in degenerative NP cells.

<p>NP cells were isolated from 63 samples of degenerative IVDs and 4 x 10⁵ cells from each sample were cultured for four weeks in collagen I scaffold. Concentration data (ELISA) of MMPs from 100 µg total protein extracts were statistically analyzed on the bases of disc degeneration grade (DDG). Box plots with whiskers min. to max. show MMP-1 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g003" target="_blank">Figure 3a</a>), MMP-2 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g003" target="_blank">Figure 3b</a>), MMP-3 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g003" target="_blank">Figure 3c</a>), MMP-7 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g003" target="_blank">Figure 3d</a>) and MMP-13 expression levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081467#pone-0081467-g003" target="_blank">Figure 3e</a>).</p

MRI showing representative images of disc degeneration grades (DDG) III, IV and V with lumbar disc herniation.

<p>MRI showing representative images of disc degeneration grades (DDG) III, IV and V with lumbar disc herniation.</p