Our previous data indicate that the expression of the PLK gene which codes for a serine/threonine kinase is restricted to proliferating cells. In Northern blot experiments PLK mRNA expression was at the limit of detection in normal lung tissue but elevated in most samples of non-small cell lung cancer (NSCLC). A very low frequency of PLK transcripts was only found in bronchiolo-alveolar carcinomas. NSCLC patients whose tumors showed moderate PLK expression survived signi®cantly longer (5 year survival rate=51.8%) than those with high levels of PLK transcripts (24.2%, P=0.001). No statistically signi®cant correlation was found between PLK mRNA expression and age, sex, TNM status, histological type or degree of dierentiation. Interestingly, the prognosis of patients in postsurgical stages I and II was correlated with PLK expression (5 year survival rates in stage I: 69.1% (moderate PLK) ± 43.5% (high PLK), P=0.03 or in stage II: 51.9% (moderate PLK) ± 9.9% (high PLK), P=0.006). These results suggest that PLK mRNA expression provides a new independent prognostic indicator for patients with NSCLC.

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signalregulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk A . cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.Neuronal development, differentiation, survival, and repair are subject to regulation by many different external signals under physiological and pathological conditions. For instance, the high affinity receptor for nerve growth factor (NGFR), 1 a receptor tyrosine kinase (RTK) also termed Trk A , is an important mediator of development, differentiation and survival of neurons (1, 2). The rat pheochromocytoma cell line PC12 is the best studied model of neuronal differentiation and survival. In these cells, nerve growth factor (NGF) causes survival upon serum-withdrawal and promotes neurite outgrowth. Activation of the epidermal growth factor receptor (EGFR), another RTK, can induce both proliferation and differentiation (3, 4); the latter response is strongly increased in EGFR-overexpressing cells (5). Activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway appears to play an important role in growth factor-mediated PC12 cell differentiation (5, 6). The mechanism of ERK1/2 activation by RTKs is well established and involves receptor autophosphorylation, recruitment of adaptor proteins such as Shc and Grb2 to the receptor, and activation of guanine nucleotide exchange factors acting on and thereby activating the small GTPase Ras. Active Ras recruits Raf kinases to the membrane, which leads to their activation and subsequent triggering of the ERK pathway (7).Studies on the pro-survival effect of NGF in PC12 cells show that activation of phosphatidylinositol 3-kinase (PI3K) is critical for its protective effect (8). Upon activation, PI3K phosphorylates membrane phosphoinositides at the D-3 position. These 3Ј-phosphorylated phospholipids act as s...

No abstract

Increased expression of the cell proliferation-associated polo-like kinase 1 (PLK1) and apoptosis-associated BCL-2 genes has been observed in different human malignancies. Inhibition of cell proliferation and reactivation of apoptosis are basic principles in anticancer therapy. The efficiency of this approach is often limited by insuf-ficient targeting and delivery of anticancer drugs into the tumors. Phosphorothioate antisense oligodeoxynucleotides (ODNs) directed against PLK1 and BCL-2 were administered systemically via the tail vein into nude mice bearing A549, MDA-MB-435, and Detroit562 xenografts. To enhance tumor-specific uptake and to reduce systemic toxicity of antisense ODNs membrane electroporation transfer was applied in vivo. Northern and Western blot analyses were used to assess PLK1 and BCL-2 expression. Tumor mass was assessed after resection of tumors. All three cell lines and corresponding xenografts expressed high levels of PLK1 and were sensitive towards antisense PLK1 treatment. Antisense BCL-2 therapy was effective in tumors expressing high levels of BCL-2, but not in A549 cells and corresponding xenografts, which express low levels of BCL-2. Administration of antisense ODNs in a dose of 5 mg/kg, twice weekly during four weeks supported by the membrane electroporation transfer, eradicated 60-100% of the xenografted tumors. Antitumor effect in BCL-2 overexpressing MDA-MB-435 cells was synergistic for BCL-2 and PLK1 combination therapy. This study provides evidence that combined systemic administration of antisense ODNs against proliferation and pro- survival associated targets and in vivo electroporation of tumors represents a promising antitumor therapeutic approach.

No abstract

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

No abstract

No abstract