Quantum dots (QDs), an important class of emerging nanomaterial, are widely anticipated to find application in many consumer and clinical products in the near future. Premarket regulatory scrutiny is, thus, an issue gaining considerable attention. Previous review papers have focused primarily on the toxicity of QDs. From the point of view of product regulation, however, parameters that determine exposure (e.g., dosage, transformation, transportation, and persistence) are just as important as inherent toxicity. We have structured our review paper according to regulatory risk assessment practices, in order to improve the utility of existing knowledge in a regulatory context. Herein, we summarize the state of academic knowledge on QDs pertaining not only to toxicity, but also their physicochemical properties, and their biological and environmental fate. We conclude this review with recommendations on how to tailor future research efforts to address the specific needs of regulators.

The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Cell cycle checkpoints are signal transduction pathways that maintain the proper order of cell cycle events (1). Several checkpoints preserve the integrity of DNA by sensing genetic anomalies and delaying progression through the cell cycle so that enough time is provided for these anomalies to be corrected. The contribution of checkpoints to human health is illustrated by a growing list of checkpoint genes that are mutated in cancer and cancer predisposition syndromes (2-8).The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a member of the checkpoint Rad family of proteins. In fission yeast, the checkpoint rad genes (rad1 ϩ , rad3 ϩ , rad9 ϩ , rad17 ϩ , rad26 ϩ , and hus1 ϩ ) are required for the S phase (DNA replication) and G 2 (DNA damage) checkpoints (9 -13). Yeasts lacking these genes fail to inactivate Cdc2 and enter premature, lethal mitosis when challenged with agents that inhibit DNA synthesis or damage DNA (14, 15). Like its yeast counterpart, hRad9 forms a ring-shaped, heterotrimeric complex with the hRad1 and hHus1 proteins (16 -18). Each member of the hRad9-hRad1-hHus1 complex (also known as the 9-1-1 complex), shares sequence homology with PCNA, 1 a homotrimer that encircles DNA and tethers DNA polymerase ␦ during DNA synthesis (19). PCNA is loaded onto DNA by the pentameric protein complex replication factor C (RFC), which is composed of one large subunit and four smaller subunits (20). In a manner analogous to PCNA and RFC, 9-1-1 is loaded onto DNA by a complex between hRad17 and the four smallest subunits of RFC (21). Since DNA damage induces hRad17-dependent association of 9-1-1 with chromatin, the 9-1-1 complex is believed to be involved in the direct recognition of DNA lesions during the initial stages of the checkpoint response (22). Also involved in this recognition are two phosphatidylinositol 3-kinase-related kinases, ATM and ATR, that regulate several cell cycle transitions and are central components of the cell checkpoint machinery (23). Even though these kinases appear to respond to different types of DNA lesions, they share a long list of common checkpoint substrates, including hRad17 (24 -26) and hRad9 (27). In fission yeast, Rad3 (which shares homology with both ATR and ATM) requires Rad9, Rad1, Hus1, and Rad17 to phosphorylate certain substrates (28). Similarly, in human cells, phosphorylation of hRad17 by ATR requires hHus1 (22). These findings support a model in which the 9-1-1 complex recruits substrates for ATM or...

ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC 50 of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.

Soluble GM-CSF receptor α subunit (sGMRα) is a soluble isoform of the GMRα that is believed to arise exclusively through alternative splicing of the GMRα gene product. The sGMRα mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRα. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRα in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRα arose exclusively through alternative splicing of the GMRα gene product, or whether it could also be generated through ectodomain shedding of GMRα, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRα (Ba/F3.GMRα). The Ba/F3.GMRα cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRα-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRα (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRα. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRα by monocytes, suggesting that shedding of GMRα by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRα is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRα secretion, and that sGMRα arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRα.

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