RESUMEN
The c-Jun NH(2)-terminal kinases (JNKs) have a role both in promoting apoptosis and tumorigenesis. The JNKs are encoded by three separate genes (JNK1, 2, and 3), which are spliced alternatively to create 10 JNK isoforms that are either M(r) 55,000 or 46,000 in size. However, the functional significance and distinct role for each splice variant remains unclear. We have noted previously that 86% of primary human glial tumors show activation of almost exclusively the M(r) 55,000 isoforms of JNK. To further study which isoforms are involved, we constructed glutathione S-transferase fusion proteins for all 10 JNK isoforms and examined kinase activity with or without the activating upstream kinase. Surprisingly, five JNK isoforms demonstrate autophosphorylation activity, and in addition, all four JNK2 isoforms (either M(r) 55,000 or 46,000) show a high basal level of substrate kinase activity in the absence of the upstream kinase, especially a M(r) 55,000 JNK2 isoform. Examination revealed autophosphorylation activity at the T-P-Y motif, which is critical for JNK activation, because a mutant lacking the dual phosphorylation sites did not show autophosphorylation or basal kinase activity. Using green fluorescence protein-JNK expression vectors, transient transfection into U87MG cells demonstrates that although the JNK1 isoforms localize predominantly to the cytoplasm, the JNK2 isoforms localize to the nucleus and are phosphorylated, confirming the constitutive activation seen in vitro. We then examined which JNK isoforms are active in glial tumors by performing two-dimensional electrophoresis. This revealed that the M(r) 55,000 isoforms of JNK2 are the principal active JNK isoforms present in tumors. Collectively, these results suggest that these constitutively active JNK isoforms play a significant role in glial tumors. Aside from epidermal growth factor receptor vIII, this is the only other kinase that has been shown to be basally active in glioma. The presence of constitutively active JNK isoforms may have implications for the design of inhibitors of the JNK pathway.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Encéfalo/enzimología , Neoplasias Encefálicas/enzimología , Activación Enzimática , Glioblastoma , Humanos , Isoenzimas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Fosforilación , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Mutación , Receptor Notch1/inmunología , Transducción de Señal/efectos de los fármacos , Células 3T3 , Animales , Especificidad de Anticuerpos/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-2 , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.