The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH 2 kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorageindependent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature. [Cancer Res 2008;68(1):81-8]

Parafibromin, a transcription factor associated with the PAF complex, is encoded by the HRPT2 gene, mutations of which cause the hyperparathyroidism-jaw tumor syndrome (OMIM145001). To elucidate the function of parafibromin, we generated conventional and conditional Hrpt2 knockout mice and found that Hrpt2 ؊/؊ mice were embryonic lethal by embryonic day 6.5 (E6.5). Controlled deletion of Hrpt2 after E8.5 resulted in apoptosis and growth retardation. Deletion of Hrpt2 in adult mice led to severe cachexia and death within 20 days. To explore the mechanism underlying the embryonic lethality and death of adult mice, mouse embryonic fibroblasts (MEFs) were cultured and Hrpt2 was deleted in vitro. Hrpt2 ؊/؊ MEFs underwent apoptosis, while Hrpt2 ؉/؉ and Hrpt2 ؉/؊ MEFs grew normally. To study the mechanism of this apoptosis, Hrpt2 ؉/؉ and Hrpt2 ؊/؊ MEFs were used in cDNA microarray, semiquantitative reverse transcription-PCR, and chromatin immunoprecipitation assays to identify genes regulated by parafibromin. These revealed that Hrpt2 expression and the parafibromin/PAF complex directly regulate genes involved in cell growth and survival, including H19, Igf1, Igf2, Igfbp4, Hmga1, Hmga2, and Hmgcs2. Thus, our results show that expression of Hrpt2 and parafibromin is pivotal in mammalian development and survival in adults and that these functions are likely mediated by the transcriptional regulation of growth factors.Loss-of-function HRPT2 mutations are associated with the autosomal dominantly inherited hyperparathyroidism-jaw tumor (HPT-JT) syndrome. Patients with HPT-JT develop parathyroid tumors, ossifying fibromas of the mandible and maxilla, uterine tumors, and renal abnormalities that may include cysts, mixed epithelial and stromal tumors, and Wilms' tumors (3). The HRPT2 gene is located on chromosome 1q31.2, and the observed loss of heterozygosity in HPT-JT-associated tumors and the identification of combined somatic and germ line HRPT2 mutations in some other HPT-JT tumors indicate that HRPT2 is a tumor suppressor gene (2, 7). In addition, HRPT2 mutations are frequently found in sporadic parathyroid carcinomas (18, 43), and expression of parafibromin, which is the 531-amino-acid protein encoded by HRPT2 (7), in parathyroid carcinomas has been reported to be lower than in normal parathyroid tissues (49). Sporadic human renal tumors, from two patients, have also been reported to have frequent allelic imbalances and to harbor HRPT2 mutations (55). These findings are consistent with a tumor suppressor role for HRPT2 and its encoded protein, parafibromin.Parafibromin shares 32% sequence similarity with the Saccharomyces cerevisiae protein Cdc73, which is a component of the yeast polymerase II-associated factor (PAF) complex, and it also has homologs in Caenorhabditis elegans, Drosophila melanogaster, and mice (7). The yeast PAF complex, which is associated with RNA polymerase II and is composed of Paf1, Ctr9, Leo1, Rtf1, and Cdc73, is involved in RNA polymerase II-mediated transcription initiation and elongation (36),...

This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

The present report describes the radioactive labeling of polar lipids in in vitro cultures of Plasmodium falciparum as well as their extraction with organic solvents and their partial characterization by chemical and enzymatic methods. All substances detected could be cleaved by alkali, suggesting that they were esters rather than sphingolipids or compounds containing alkyl groups. Dolichol-cycle intermediates were not detected. Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine were labeled by fatty acids and inositol or ethanolamine, respectively, confirming their de novo synthesis by the parasite. Metabolic labeling with glucosamine and cleavage by phosphatidylinositol-specific phospholipase C provided evidence of the formation of N-acetyl-glucosaminyl-phosphatidylinositol, an obligate precursor in the biosynthesis of glycosylphosphatidylinositol membrane anchors of proteins.

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