Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end joining.

The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18(-/-), RNF8(-/-) and RAD18(-/-)/RNF8(-/-) clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13(-/-)cells, they are more sensitive to camptothecin and olaparib than UBC13(-/-)cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18(-/-) and RNF8(-/-) cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy-induced DSB sites.

Hypoxia as a target for combined modality treatments.

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents.

There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells. To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells. Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins. The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages. Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis. Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.

Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine.

Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.

Alternatively spliced CD44 isoforms containing exon v10 promote cellular adhesion through the recognition of chondroitin sulfate-modified CD44.

Correlations have been noted between the expression of certain alternatively spliced CD44 isoforms and the metastatic propensity of various histologically distinct tumor cell types. The precise mechanism by which particular CD44 isoforms contribute to the metastatic process is, however, unclear. In the present study we demonstrate that CD44R2, a CD44 isoform highly expressed on activated and transformed hemopoietic cells, can recognize and bind a common determinant present on CD44H and CD44R1. Importantly, CD44H lacked this activity. Pretreatment of TIL1 cells expressing CD44H or CD44R1 with chondroitinase ABC inhibited adhesion to CD44R2, suggesting that the unique inserted region present within the CD44R2 molecule, encoded by exon v10, mediates cell adhesion by potentiating the recognition of chondroitin sulfate moieties presented in association with other CD44 molecules. These data help explain the differential involvement of v10-containing CD44 isoforms in tumor metastasis.

Identification and characterization of CD44RC, a novel alternatively spliced soluble CD44 isoform that can potentiate the hyaluronan binding activity of cell surface CD44.

Soluble CD44 proteins generated by proteolytic cleavage or aberrant intron retention have been shown to antagonize the ligand binding activity of the corresponding cell surface receptor, inducing apoptosis and inhibiting tumor growth. Interestingly, such findings appear to contradict recent studies demonstrating a correlation between the presence of high levels of soluble CD44 in the serum of cancer patients and poor prognosis. In the present study, we report the cloning of a novel, naturally occurring, differentially expressed, soluble CD44 isoform, designated CD44RC, which, in contrast to previously described soluble CD44 proteins, can dramatically enhance the hyaluronan binding activity of cell surface CD44. Sequence analysis suggests that CD44RC is generated by an alternative splicing event in which the 3' end of CD44 exon 2 is spliced into an internal splice acceptor site present within exon 18, altering reading frame and giving rise to a soluble protein with a unique COOH terminus. Functional studies suggest that CD44RC enhances hyaluronan binding by adhering to chondroitin sulfate side-chains attached to cell surface CD44, generating a multivalent complex with increased avidity for hyaluronan.

Molecular mechanisms regulating the hyaluronan binding activity of the adhesion protein CD44.

In the present study, we describe the isolation and characterization of a cDNA clone designated B6F1.3, that appears to 'activate' the hyaluronan-binding capacity of CD44 upon transfection into the murine fibroblastoid cell line MOP8. Sequence analysis indicates that the putative regulatory molecule encoded by this clone is identical to the murine interleukin-2 receptor gamma chain (mIL-2R gamma), a recently described type 1 transmembrane protein that constitutes an integral component of the cell surface receptors that bind a number of cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and perhaps also IL-13. Mutations in this molecule have been shown to be responsible for X-linked severe combined immunodeficiency (XSCID) in humans. With the exception of bone marrow, the mIL-2R gamma chain was found to be expressed at high levels on all hemopoietic cell lines and tissue types examined. Non-hemopoietic tissues are generally negative. FACS analysis and Western blot analysis indicated respectively that B6F1.3 does not mediate its effects by upregulating the expression of CD44 or by altering the alternative splicing of the molecule. Removal of the cytoplasmic tail of the mIL-2R gamma chain, including a Src homology region 2 (SH2) subdomain, abolished its ability to enhance CD44-mediated binding to hyaluronan suggesting the involvement of signal transduction events triggered via the cytoplasmic domain in the 'activation' process. Determining whether activating molecules such as B6F1.3 are co-expressed within tumor cells may help improve the potential value of CD44 as a diagnostic marker of metastatic disease.

Adhesive interactions between alternatively spliced CD44 isoforms.

Alternative splicing of a series of 10 contiguous exons present within the CD44 gene can generate a large number of differentially expressed CD44 isoforms that contain additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule proximal to the membrane spanning domain. Although distinct functions have been ascribed to certain of these isoforms, the effect of particular inserted domains on the ligand-binding specificity of the CD44 molecule remains unclear. In the present study, we demonstrate that while CD44H, the major CD44 isoform expressed on resting hemopoietic cells, and CD44R1, an alternatively spliced isoform present on transformed epithelial cells and certain activated and/or malignant hemopoietic cell types, can both bind avidly to hyaluronan, only CD44R1 can promote homotypic cellular aggregation when expressed in the CD44-negative murine lymphoma cell line TIL1. Experiments in which TIL1 cells transduced with different CD44 isoforms were tested for their ability to adhere to one another or to COS7 cells transfected with CD44R1, indicated that CD44R1 can recognize and bind a common determinant present on both CD44H and CD44R1. Monoclonal antibody blocking studies suggest further, that the determinant recognized by CD44R1 is located in a region of the CD44 molecule distinct from that involved in hyaluronan binding.

Ligand binding specificity of alternatively spliced CD44 isoforms. Recognition and binding of hyaluronan by CD44R1.

CD44 species of widely differing molecular mass have been identified on various normal and/or transformed cells. Recent studies have demonstrated that much of this heterogeneity is produced as a result of the alternative splicing of a series of 10 exons present within the CD44 gene generating a large number of CD44 isoforms containing additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule. At present, the effect of such insertions on the ligand binding specificity of CD44 remains unclear. CD44H, the major CD44 isoform expressed by most resting cell types, has been shown to function as a receptor for the glycosaminoglycan hyaluronan. In contrast, CD44E, the major isoform expressed by the colon carcinoma cell line HT29, which contains a 132-amino acid insert, is unable to recognize and bind this ligand. In the present study we demonstrate that CD44R1, an isoform isolated from the myelomonocytic cell line KG1a, that differs from CD44E by just 3 amino acid substitutions, is fully capable of mediating the attachment of transfected COS7 cells to hyaluronan-coated plastic. In order to confirm that such binding was directly mediated by the introduced CD44 species, chimeric proteins containing the entire extracellular domain of CD44H or CD44R1 fused in-frame to human bone/liver/kidney alkaline phosphatase were prepared and tested for their ability to bind hyaluronan-coated plastic. Both fusion proteins bound equally well to hyaluronan and in each case their attachment could be readily inhibited by monoclonal antibodies directed against the hyaluronan-binding domain of CD44. These data indicate that the 132-amino acid insert present within the extracellular domain of CD44R1 does not interfere with the hyaluronan binding function of the molecule. Since CD44E contains an identically sized insert but is unable to bind hyaluronan, it is likely that mutation of one or more of the 3 amino acid residues that differ between CD44E and CD44R1 is responsible for the altered functional activity of this particular molecule.