Radiation-hypersensitive cancer patients do not manifest protein expression abnormalities in components of the nonhomologous end-joining (NHEJ) pathway.

Radiation therapy (RT) is utilised for the treatment of around half of all oncology patients during the course of their illness. Despite great clinical progress in the rational deployment of RT, the underlying molecular basis for its efficacy and toxicity are currently imperfectly understood. In this study, we took a biochemical approach to evaluate the potential role of key ionising radiation repair proteins in the treatment outcomes of patients with severe acute or late RT side effects. Lymphoblastoid cell lines were established from blood samples from 36 radiosensitive cases and a number of controls (the latter had had RT but did not develop significant toxicity). The expression level and migration of key proteins from the nonhomologous end-joining (NHEJ) pathway was evaluated by Western blot analysis on cases and controls. We did not observe any abnormalities in expression level or migration pattern of the following NHEJ proteins in radiosensitive cancer cases: Ku70, Ku80, XRCC4, DNA Ligase IV. These important negative results provide evidence that mutations that affect protein expression of these NHEJ components are unlikely to underlie clinical radiation sensitivity.

Characterization of a novel human cell-cycle-regulated homologue of Drosophila dlg1.

Cell cycle defects have been associated with the process of carcinogenesis in many studies. Here we report the cloning and analysis of the novel gene KIAA0008 (GenBank acc. no. D13633). Chromosomal localization experiments assigned the gene to chromosome 14q22-q23. The mRNA transcript was found to be cell cycle regulated, expressed at S-phase, and maintained at both G2-and M-phases. In situ hybridization showed expression in proliferating colon and breast (tumor) tissues. Structurally, KIAA0008 shares homology with the Drosophila melanogaster discs large-1 (dlg1) tumor suppressor gene and membrane-associated guanylate kinase protein family members. The potential involvement of KIAA0008 in cell proliferation is discussed, along with its sequence identity and tissue distribution.

Functional abnormalities in protein tyrosine phosphatase epsilon-deficient macrophages.

Protein tyrosine phosphatase epsilon (PTP epsilon)-deficient mice were generated by targeted deletion of exons 3, 4, and 5 of the Ptpre gene. Mice homozygous for this deletion (Ptpre(Delta3-5)) were fertile, bred and developed normally and exhibited no overt phenotype. However, closer examination of the function of macrophages from these mice revealed a defect in the regulation of the respiratory burst. While bacterial lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNFalpha) were able to prime bone marrow-derived macrophages (BMM) from wild type (Ptpre(+)) macrophages for an enhanced respiratory burst, they were unable to do so in macrophages from PTP epsilon-deficient mice. PTP epsilon-deficient BMM also had abnormalities in cytokine production with a reduced ability to produce TNFalpha and enhanced IL-10 production in response to challenge with LPS. These findings suggest an important role for PTP epsilon in the control of macrophage function.

Phosphotyrosine phosphatase activity in the macrophage is enhanced by lipopolysaccharide, tumor necrosis factor alpha, and granulocyte/macrophage-colony stimulating factor: correlation with priming of the respiratory burst.

Tyrosine phosphorylation is now recognised as a key event in the activation of the macrophage respiratory burst. Since vanadate, a phosphotyrosine phosphatase (PTP) inhibitor is able to enhance the respiratory burst, we proposed that agents which prime the macrophage for enhance respiratory burst activity may do so by suppressing cellular PTP activity. The level of PTP activity in murine bone marrow-derived macrophages (BMM) was assessed by the ability of cell lysates to dephosphorylate 32P-labelled RR-src peptide. In contrast to our hypothesis, pretreatment of BMM with bacterial lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or granulocyte/macrophage-colony stimulating factor (GMCSF), agents which prime for enhanced respiratory burst activity, was found to dramatically increase the level of cellular PTP activity. The time-course for this increase correlated well with the time course of priming by these agents. In addition, colony stimulating factor-1, a cytokine which does not prime the macrophage respiratory burst, did not enhance PTP levels. The physiological relevance of the increased PTP activity was further supported by confirming it was active against endogenous tyrosine phosphorylated substrates. Interestingly, phorbol myristate acetate and zymosan, agents which trigger the macrophage respiratory burst, were found to inhibit the PTP activity of BMM. Our results demonstrate the regulation of cellular PTP activity by priming agents and further highlight the importance of tyrosine phosphorylation and dephosphorylation events in the regulation of macrophage function.

Tyrosine phosphorylation: a signal for the activation of the phagocyte respiratory burst.

The respiratory burst of the phagocytic cell is a unique function which provides the cell with a series of reactive oxygen intermediates which it can use to kill ingested microorganisms. The complex signal transduction pathways responsible for regulating the respiratory burst are yet to be fully elucidated. Protein tyrosine kinases are now recognised as being critical components in the regulatory pathways controlling many cellular functions. In this report we review the evidence implicating tyrosine phosphorylation as an important signal for the activation of the phagocyte respiratory burst.