Despite a substantial understanding of the factors regulating oligodendrocyte differentiation, the signaling mechanisms involved in this process are not well-understood. This study elaborates on the findings (Bhat NR, Zhang P (1997) FASEB J 11:A925; Baron W, Metz B, Bansal R, Hoekstra D, de Vries H (2000) Mol Cell Neurosci 15:314-329) of a role for p38 MAP kinase signaling in oligodendrocyte differentiation and myelin gene expression. When proliferating oligodendrocyte progenitors were switched to a growth factor-free differentiation medium, there was a rapid activation of p38 kinase that correlated with an increased phosphorylation of CREB, a down-stream target and a factor involved in oligodendrocyte differentiation. Addition of forskolin, a known inducer of intracellular c-AMP and of oligodendrocyte differentiation, also stimulated CREB phosphorylation in a p38 kinase dependent way. Pharmacological inhibition of p38 interfered with the morphological and antigenic changes associated with differentiating oligodendrocytes as well as with the developmental and forskolin-induced expression of myelin basic protein, thereby supporting an essential role for p38 MAPK pathway in oligodendrocyte differentiation.

Tumor progression locus 2 (Tpl2)/Cot kinase is a newer member of MAP3K family that is now known for its essential role in TNFα expression in macrophages, but its proinflammatory signaling, if any, in glia is unknown. When cultures of murine microglia and astrocytes were exposed to lipopolysaccharide, there was a rapid activation (i.e., phosphorylation) of Tpl2 in parallel to the activation of down-stream effector MAPKs i.e., ERK, p38 MAPK and JNK. Pre-incubation of the cultures with a Tpl2 inhibitor selectively suppressed the activation of the primary down-stream target i.e., ERK relative to p38 MAPK and JNK. That Tpl2 activation was functionally involved in glial inflammatory response was indicated by a reduced release of the cytokines i.e., TNFα and the expression of inducible nitric oxide synthase (iNOS) in the presence of the kinase inhibitor. Further, overexpression of a wild-type Tpl2 construct in C-6 glia resulted in an enhanced transcriptional activation of iNOS while transfection with a dominant negative form of Tpl-2 had the opposite effect. The findings assign an important proinflammatory signaling function for Tpl2 pathway in glial cells.

In this report, we describe the development and characterization of an anti-ME-180 cervical cancer-specific epidermal growth factor (EGF) receptor monoclonal antibody (MAb). This MAb, 6C7, specifically binds to ME-180 cervical cancer cells and not to normal cervical epithelial cells. By immunoaffinity chromatography, we have shown that the 6C7 antibody binds to a 205-kDa protein. Subsequent mass spectrometry sequencing analysis identified this protein as an EGF receptor. In addition, treatment of the ME-180 EGF receptor with N- and O-linked glycosidases indicated that this antibody binds to the carbohydrate portion of the glycoprotein. Moreover, Western blotting analysis with an anti-EGF receptor antibody indicated that this protein is present in abundance in all cervical cancer cell lines, including ME-180, HeLa, Ca Ski, HT-3, SiHa, and Hs 588.T. However, the 6C7 antibody only binds to the EGF receptor from ME-180 cells, suggesting that this protein is differentially glycosylated in ME-180 cells, compared to other cervical cancer cell lines. Finally, we have shown that this antibody could selectively block EGF-mediated cell proliferation in ME-180 cells but not in HeLa cells. Overall, our study suggests that the differentially glycosylated EGF receptor could potentially serve as a unique target for the immunotherapeutic treatment of cervical cancer.

In this report, we describe the development and characterization of monoclonal antibodies against the surface antigens of cervical cancer cells. Using HeLa cervical carcinoma cells as the immunogen, we have developed a number of antibodies that specifically label cervical cancer cells but not normal cervical epithelial cells. These antibodies displayed differential reactivity towards various cervical cancer cell lines as determined by immunofluorescence labeling and western blotting analyses. One of these antibodies, 13G4, which showed the strongest labeling to HeLa cells and has the widest range of reactivity to other cervical cancer cell lines, was extensively characterized. By immunoaffinity chromatography, we purified a 90-kDa protein that appears to be the principal target recognized by this antibody. This protein was subsequently identified as decay accelerating factor (DAF) or CD55 by the mass spec sequencing analysis of the tryptic peptides derived from this protein. Digestion of HeLa DAF with glycosidases that removed its N- and O-linked carbohydrates has revealed that the 13G4 antibody binds to the peptide portion of this glycoprotein. Overall, our approach of generating and characterizing monoclonal antibodies directed against the surface antigens of cervical cancer cells serves as a stepping stone towards the eventual development of a unique panel of monoclonal antibodies that could potentially be used for the detection and therapeutic treatment of cervical cancer.