Haematopoietic stem cell donor registries: World Marrow Donor Association recommendations for evaluation of donor health.

The ability to identify unrelated haematopoietic stem cell donors in one country for recipients in another country requires cooperation and standardization in many areas. The donor assessment and testing are very important issues affecting quality and safety of donation. This special report details the World Marrow Donor Association's recommended procedures regarding the medical evaluation of donors, with the intent to protect the volunteer from the risk to damage his health and to offer the recipient the appropriate quality of stem cells. This document describes criteria for permanent or temporary deferral, guidelines for risk evaluation of infectious disease, examples of conditions requiring assessment and questionnaires designed to elicit relevant information about a donor's medical history and general health.

Informed consent--suggested procedures for informed consent for unrelated haematopoietic stem cell donors at various stages of recruitment, donor evaluation, and donor workup.

The Ethics Working Group of the World Marrow Donor Association (WMDA) was established to address the increasing and complex number of ethical issues surrounding unrelated haematopoietic stem cell donation where the selected donor and recipient reside in different countries. This paper considers the topic of informed donor consent, but recognises that the recommendations contained within the paper may be subject to cultural variances in interpretation, and to adjustment to meet the legal requirements of individual countries. Nevertheless, the extent of international cooperation establishes sufficient common denominators for the recommendations to be widely adhered to in the interests of best practice.

Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae.

Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells. Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown. Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents. We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents. Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents. Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen.

Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains.

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Mating in Saccharomyces cerevisiae: the role of the pheromone signal transduction pathway in the chemotropic response to pheromone.

The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the beta or gamma subunit (ste4 and ste18) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.

RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage.

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9 delta, rad 17 delta, and rad24 delta. Furthermore, the triple rad9 delta rad17 delta rad24 delta mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1 delta, mag1 delta, apn1 delta, rev3 delta, rad18 delta, rad16 delta, dun1-delta 100, sad4-1, tel1 delta, rad26 delta, rad51 delta, rad52-1, rad54 delta, rad14 delta, rad1 delta, pol30-46, pol30-52, mad3 delta, pds1 delta/esp2 delta, pms1 delta, mlh1 delta, and msh2 delta are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.

Cell cycle control and cancer.

Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.

S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination.

Wild-type S. cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone. However, some mutants that are supersensitive to pheromone lack this ability to discriminate. In this study, we provide evidence for a novel role of alpha pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway. Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination. Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.

Courtship in S. cerevisiae: both cell types choose mating partners by responding to the strongest pheromone signal.

We demonstrate that during the courtship stage of conjugation, S. cerevisiae a cells choose the alpha cell producing the highest level of pheromone from among potential mating partners. From this result and that for alpha cells we conclude that both a and alpha cells act as signaling cells during courtship, that both cell types respond by discriminating different levels of signal, and that the signals are the mating pheromones. Responding cells that are supersensitive to signal fail to discriminate pheromone-producing from nonproducing cells to an extent that depends on their degree of supersensitivity. We propose that partner selection in S. cerevisiae results from polarized morphogenesis of a responding cell in the direction of highest pheromone concentration and that cells defective in discriminating this gradient execute a default pathway in which an adjacent cell is selected at random.

Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating.

During conjugation in Saccharomyces cerevisiae, two cells of opposite mating type (MATa and MAT alpha) fuse to form a diploid zygote. Conjugation requires that each cell locate an appropriate mating partner. To investigate how yeast cells select a mating partner, we developed a competition mating assay in which wild-type MAT alpha cells have a choice of two MATa cell mating partners. We first demonstrated that sterile MAT alpha 1 cells (expressing no a- or alpha-specific gene products) do not compete with fertile MATa cells in the assay; hence, wild-type MATa and MAT alpha cells can efficiently locate an appropriate mating partner. Second, we showed that a MATa strain need not be fertile to compete with a fertile MATa strain in the assay. This result defines an early step in conjugation, which we term courtship. We showed that the ability to agglutinate is not necessary in MATa cells for courtship but that production of a-pheromone and response to alpha-pheromone are necessary. Thus, MATa cells must not only transmit but must also receive and then respond to information for effective courtship; hence, there is a "conversation" between the courting cells. We showed that the only alpha-pheromone-induced response necessary in MATa cells for courtship is production of a-pheromone. In all cases tested, a strain producing a higher level of a-pheromone was more proficient in courtship than one producing a lower level. We propose that during courtship, a MAT alpha cell selects the adjacent MATa cell producing the highest level of a-pheromone.

The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone.

STE2 encodes a component of the S. cerevisiae alpha-pheromone receptor that is essential for induction of physiological changes associated with mating. Analysis of C-terminal truncation mutants of STE2 demonstrated that the essential sequences for ligand binding and signal transduction are included within a region containing seven putative transmembrane domains. However, truncation of the C-terminal 105 amino acids of the receptor resulted in a 4- to 5-fold increase in cell-surface pheromone binding sites, a 10-fold increase in pheromone sensitivity, a defect in recovery of cell division after pheromone treatment, and a defect in pheromone-induced morphogenesis. Overproduction of STE2 resulted in about a 6-fold increase in alpha-pheromone binding capacity but did not produce the other phenotypes associated with the ste2-T326 mutant receptor. We conclude that the C-terminus of the receptor is responsible for one aspect of cellular adaptation to pheromone that is distinct from adaptation controlled by the SST2 gene, for decreasing the stability of the receptor, and for some aspect of cellular morphogenesis.