ABSTRACT
To identify genes that could serve as targets for novel cancer therapeutics, we used a bioinformatic analysis of microarray data comparing gene expression between normal and tumor-derived primary human tissues. From this approach, we have found that maternal embryonic leucine zipper kinase (Melk), a member of the AMP serine/threonine kinase family, exhibits multiple features consistent with the potential utility of this gene as an anticancer target. An oligonucleotide microarray analysis of multiple human tumor samples and cell lines suggests that Melk expression is frequently elevated in cancer relative to normal tissues, a pattern confirmed by quantitative reverse transcription-PCR and Western blotting of selected primary tumor samples. In situ hybridization localized Melk expression to malignant epithelial cells in 96%, 23%, and 13% of colorectal, lung, and ovarian tissue tumor samples, respectively. Expression of this gene is also elevated in spontaneous tumors derived from the ApcMin and Apc1638N murine models of intestinal tumorigenesis. To begin addressing whether Melk is relevant for tumorigenesis, RNA interference-mediated silencing within human and murine tumor cell lines was done. We show that Melk knockdown decreases proliferation and anchorage-independent growth in vitro as well as tumor growth in a xenograft model. Together, these results suggest that Melk may provide a growth advantage for neoplastic cells and, therefore, inactivation may be therapeutically beneficial.
Subject(s)
Neoplasms/enzymology , Protein Serine-Threonine Kinases/biosynthesis , 3T3 Cells , Amino Acid Sequence , Animals , Computational Biology , HeLa Cells , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Neoplasms/genetics , Neoplasms/therapy , Oligonucleotide Array Sequence Analysis , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , RNA Interference , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: To investigate the role of nitric oxide (NO) in basal and cytokine induced cartilage matrix breakdown and synthesis across different species and in chondrocytes cultured as isolated cells or as tissue explants. METHODS: Articular cartilage from bovine, porcine, or human joints was cultured as explants in serum-free media. Explants or monolayer cultures of primary chondrocytes were treated with cytokines in the absence or presence of inhibitors [antibodies to leukemia inhibitory factor (anti-LIF) or tumor necrosis factor-alpha, dexamethasone, or inhibitors of aggrecanase or NO synthase]. NO production and matrix breakdown and synthesis were measured. RESULTS: At low concentrations, a novel interleukin 17 (IL-17) family member induced matrix breakdown without altering NO production. Treatment of articular cartilage explants with dexamethasone or anti-LIF blocked NO production by IL-17, but not by IL-1alpha. Inhibition of NO production in cytokine treated cartilage explants enhanced matrix breakdown and partially overcame suppression of matrix synthesis. In isolated chondrocytes, inhibition of NO production decreased expression of gelatinase and increased expression of stromelysin. CONCLUSION: Endogenous NO serves a dual function in cartilage: to protect the tissue from matrix breakdown and to mediate suppression of proteoglycan synthesis by cytokines. Despite the similarities in biological function between IL-I and IL-17, their downstream signaling pathways are distinct and appear to be affected by extracellular matrix degradation.
