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The haematopoietic system is a complex organised tissue with a hierarchical structure. Identification of organisational pathways within the haematopoietic system is relevant for a better understanding of haematopoiesis in health and disease. We have analysed numerous haematopoietic parameters in two panels of a total of 157 genetically distinct B6AKRF2 mice, derived from an intercross between AKR and C57Bl/6 mice, strains known to differ in various stem cell traits. The major objective of our study was to assess the extent to which various haematopoietic parameters, such as stem cell numbers, progenitor cell cycling, progenitor cell mobilisation and neutrophil numbers in blood and bone marrow are coregulated. The genotypes of these mice were used to search for genetic loci that regulate these parameters. We found significant quantitative trait loci (QTL) associated with the number of stem cells (CAFC-35) in the bone marrow and the number of neutrophils in the blood. However, most haematopoietic parameters appeared to be controlled by non-heritable (epigenetic) factors, or by multiple QTLs. Our study reveals striking differences in structure of the haematopoietic hierarchy between individual mice. Surprisingly, stem and progenitor cell pool size and proliferation rate, as well as peripheral blood cell counts are all independently regulated.

The differentiation of hematopoietic stem cells into mature blood cell lineages is tightly regulated. Here we report that CD27, which is expressed on stem and early progenitor cells in bone marrow, can be important in this process. Deletion of CD27 increased the myeloid colony-forming potential of stem and early progenitor cells and enhanced B lymphoid reconstitutive capacity in competitive transplantation experiments. Conversely, stimulation of CD27(+) progenitor cells with CD70, the unique ligand for CD27, inhibited colony-forming potential in vitro and lymphocyte outgrowth in vivo. As CD70 is expressed only on activated immune cells, we suggest that CD27 triggering on early progenitor cells provides a negative feedback signal to leukocyte differentiation during immune activation.

Controlled ovarian hyperstimulation (COH) used in IVF produces lower implantation rates per embryo transferred compared to natural cycles utilized in ovum donation, suggesting a suboptimal endometrial development. Endometrial receptivity has recently been investigated in natural menstrual cycles with the aid of microarray technology. The aim of this study is to investigate the impact of COH using urinary gonadotrophins with a long protocol with GnRH agonists without progesterone supplementation (similar to the natural cycle) on endometrial gene expression profiles during the window of implantation by comparing the profiles at day hCG + 7 of COH versus LH + 7 of a previous natural cycle in the same women. For this purpose we have used microarray technology by Affymetrix (GeneChip HG_U133A), which allows more than 22,000 genes to be tested simultaneously. Results were validated by semi-quantitative PCR and quantitative PCR experiments. We found that more than 200 genes showed a differential expression of more than 3-fold when COH and normal cycles were compared at hCG + 7 versus LH + 7. We simultaneously re-analysed the LH + 2 versus LH + 7 endometrial gene expression profiles in previous natural cycles in the same subject using this specific GeneChip, the results obtained were consistent with our own published results. This is the first time that gene expression profiles of the endometrium during COH are reported. The large degree of gene expression disturbance is surprising and highlights the need for further efforts to optimize COH protocols.

Adult somatic stem cells possess extensive self-renewal capacity, as their primary role is to replenish aged and functionally impaired tissues. We have previously shown that the stem cell pool in short-lived DBA/2 (D2) mice is reduced during aging, in contrast to long-lived C57BL/6 (B6) mice. This suggests the existence of a genetically determined mitotic clock operating in stem cells, which possibly limits organismal aging. In the study reported here, unfractionated bone marrow (BM) cells or highly purified Lin(-)Sca-1(+)c-kit(+) (LSK) cells were serially transplanted in lethally irradiated D2 and B6 mice. In both strains, serial transplantation resulted in a substantial loss of stem cell activity. However, as we estimate that in B6 mice, the maximum number of population doublings of primitive cells is approximately 30, in D2 mice this is only approximately 20, resulting in a 1,000-fold difference in expansion potential, irrespective of whether whole bone marrow or purified hematopoietic stem cells (HSCs) were transplanted. Interestingly, recipients reconstituted with serially transplanted BM cells were able to accept a freshly isolated graft without any further conditioning. Finally, we show that whereas transplantation of BM cells into healthy, nonconditioned, young B6 recipients does not lead to engraftment, young BM cells do engraft and provide multilineage reconstitution in nonirradiated aged mice. Our data clearly establish the relevance of an intrinsic, genetically controlled program associated with impaired stem cell functioning during aging.

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 +/- 61 vs 290 +/- 64, P <.05; G-CSF: 1925 +/- 1216 vs 3371 +/- 1214, P <.05; FL: 562 +/- 213 vs 1068 +/- 528, P <.05). Treatment with ciprofloxacin eliminated only aerobic Gram-negative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 +/- 66 vs 538 +/- 216, P <.05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 +/- 182, IL-8 germ free: 157 +/- 53, P <.05). Finally, we performed reconstitution experiments adding Escherichia coli-derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8-induced mobilization (67 +/- 28 vs 190 +/- 98.1, P <.01). Our results indicate that endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization.

Numerous assays exist that measure the function of stem cells. In this article, we review in detail the history and future of existing stem cell assays. Hematopoietic stem cells (HSCs) are historically the most well studied, but new developments in stem cell research, including the claim of stem cell plasticity, have caused controversies related to technical issues, as well as to semantics. Stem cell research requires proper definitions, and utilization of stem cell assays, especially since research on non-HSCs that lack solid stem cell assays, is rapidly evolving. These emerging fields may benefit from what has been learned from HSC assays: most important, that the true potential of stem cells can only be assessed retrospectively. This also relates to new developments in HSC research, when limiting numbers of in vitro-manipulated stem cells are transplanted. The most conflicting results arise when cells express stem cell characteristics in one assay but not in another. Should we adjust our definition of a stem cell? If so, when do we decide a claim of stem cell activity to be justified? We therefore recommend using multiple stem cell assays, preferably at least one in vivo assay. These assays should measure functionality of the putative stem cell population.

Objective: Autologous bone marrow transplantation in cancer patients is often preceded by multiple cycles of chemotherapy. In this study, we assessed in a mouse model whether stem cells were affected by prior chemotherapy., Methods: Donor mice were treated with three consecutive injections of 150 mg/kg 5-fluorouracil (5-FU). Peripheral blood counts were allowed to recover before the subsequent dose of 5-FU was given. Mice recovered from three doses of 5-FU and showed normal steady-state hematopoiesis. Bone marrow cells from these mice were mixed with congenic competitor cells and transplanted into lethally irradiated recipients., Results: Although in vivo homing of cells from these mice was not impaired, donor leukocyte contribution steadily decreased posttransplantation. In contrast to in vivo homing, both in vitro migration toward stromal-derived factor (SDF)-1 and the average CXC chemokine receptor-4 (CXCR4) expression were lower in 5-FU-treated cells. Moderate reductions in L-selectin and CD11a expression were observed on stem cells of 5-FU-treated mice. CD43, CD44, CD49d, and CD49e were normally expressed and could thus not explain the reduced engraftment of these cells., Conclusion: We therefore conclude that 5-FU either directly damages stem cells or that the replicative stress induced by 5-FU causes a decline in stem cell reconstitution potential resulting in lower chimerism levels posttransplantation, that declines in time.

In humans, embryonic implantation and reproduction depends on the interaction of the embryo with the receptive endometrium. To gain a global molecular understanding of human endometrial receptivity, we compared gene expression profiles of pre-receptive (day LH+2) versus receptive (LH+7) endometria obtained from the same fertile woman (n = 5) in the same menstrual cycle in five independent experiments. Biopsies were analysed using the Affymetrix HG-U95A array, a DNA chip containing approximately 12,000 genes. Using the pre-defined criteria of a fold change >/=3 in at least four out of five women, we identified 211 regulated genes. Of these, 153 were up-regulated at LH+7 versus LH+2, whereas 58 were down-regulated. Amongst these 211 regulated genes, we identified genes that were known to play a role in the development of a receptive endometrium, and genes for which a role in endometrial receptivity, or even endometrial expression, has not been previously described. Validation of array data was accomplished by mRNA quantification by real time quantitative fluorescent PCR (Q-PCR) of three up-regulated [glutathione peroxidase 3 (GPx-3), claudin 4 (claudin-4) and solute carrier family 1 member 1 (SLC1A1)] genes in independent LH+2 versus LH+7 endometrial samples from fertile women (n = 3) and the three up-regulated genes throughout the menstrual cycle (n = 15). Human claudin-4 peaks specifically during the implantation window, whereas GPx-3 and SLC1A1 showed highest expression in the late secretory phase. In-situ hybridization (ISH) experiments showed that GPx-3 and SLC1A1 expression was restricted to glandular and luminal epithelial cells during the mid- and late luteal phase. The present work adds new and important data in this field, and highlights the complexity of studying endometrial receptivity even using global gene-expression analysis.

The role of fibroblast growth factors and their receptors (FGFRs) in the regulation of normal hematopoietic stem cells is unknown. Here we show that, in mouse bone marrow, long-term repopulating stem cells are found exclusively in the FGFR(+) cell fraction. During differentiation toward committed progenitors, stem cells show loss of FGFR expression. Prolonged culture of bone marrow cells in serum-free medium supplemented with only FGF-1 resulted in robust expansion of multilineage, serially transplantable, long-term repopulating hematopoietic stem cells. Thus, we have identified a simple method of generating large numbers of rapidly engrafting stem cells that have not been genetically manipulated. Our results show that the multipotential properties of stem cells are dependent on signaling through FGF receptors and that FGF-1 plays an important role in hematopoietic stem cell homeostasis.

The beta 2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34(+) cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti-LFA-1 antibodies completely prevent the IL-8-induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-beta 2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the alpha chain of LFA-1 or against the alpha chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area-forming cells in vitro was significantly higher after mobilization with anti-LFA-1 antibodies followed by 5 microg G-CSF for 5 days than with G-CSF alone (119 +/- 34 days vs 17 +/- 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF-induced mobilization, suggesting that the enhancing effect required an interaction of the beta 2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1-deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1-mediated adhesion.

The CXC chemokine interleukin-8 (IL-8/CXCL8) induces rapid mobilization of hematopoietic progenitor cells (HPCs). Previously we showed that mobilization could be prevented completely in mice by pretreatment with neutralizing antibodies against the beta2-integrin LFA-1 (CD11a). In addition, murine HPCs do not express LFA-1, indicating that mobilization requires a population of accessory cells. Here we show that polymorphonuclear cells (PMNs) serve as key regulators in IL-8-induced HPC mobilization. The role of PMNs was studied in mice rendered neutropenic by administration of a single injection of antineutrophil antibodies. Absolute neutropenia was observed up to 3-5 days with a rebound neutrophilia at day 7. The IL-8-induced mobilizing capacity was reduced significantly during the neutropenic phase, reappeared with recurrence of the PMNs, and was increased proportionally during the neutrophilic phase. In neutropenic mice, the IL-8-induced mobilizing capacity was restored by the infusion of purified PMNs but not by infusion of mononuclear cells. Circulating metalloproteinase gelatinase B (MMP-9) levels were detectable only in neutropenic animals treated with PMNs in combination with IL-8, showing that in vivo activated PMNs are required for the restoration of mobilization. However, IL-8-induced mobilization was not affected in MMP-9-deficient mice, indicating that MMP-9 is not indispensable for mobilization. These data demonstrate that IL-8-induced mobilization of HPCs requires the in vivo activation of circulating PMNs.

Various techniques are available for distinguishing donor from host cells evaluating the efficacy of conditioning regimen for experimental bone marrow transplantation (BMT). Techniques include the use of extracellular immunological markers, such as Ly5 (CD45), and intracellular biochemical markers, such as glucose-phosphate-isomerase (Gpi). Because Ly5 is an extracellular protein, the disparity between donor (Ly5.1) and host (Ly5.2) antigens may induce a weak immune response whereas with Gpi, no immune response is expected. This difference may be of particular concern in experimental transplantation approaches that use minimal conditioning such as low-dose total body irradiation (TBI). Such mild conditioning may not induce the immunosuppression required to overcome host rejection of Ly5 disparate cells. To compare the relative engraftment of Ly5.1 and Gpi-1(a) donor marrow, B6 (Gpi-1(b)/Ly5.2) mice were irradiated with low-level TBI (0-6 Gy) and transplanted with several bone marrow (BM) doses (2 x 10(6)-5 x 10(7) cells). At 8, 26, and 52 weeks post-BMT, the level of donor engraftment was measured using flow cytometry (Ly5) or Gpi-electrophoresis. Lower engraftment levels were found in mice transplanted with Ly5 congenic BM in groups given low-dose TBI (< or = 4 Gy) and/or low doses of BM cells (BMC) (2 x 10(6)). However, when higher TBI or BMC doses were used, similar engraftment levels were found, suggesting sufficient immune suppression to allow equal engraftment of both sources of BM. These data suggest that even a minor phenotypic disparity between donor and host, such as Ly5, may necessitate high-dose TBI to prevent rejection. The combination of low-dose TBI or other nonmyeloablative conditioning strategies with small numbers of BMC may lead to reduced engraftment when extracellular immunological markers such as Ly5 are used for transplantation studies. Therefore, small immunological differences must be considered when using the Ly5 marker for engraftment.

It has been suggested that mature neutrophils may play an essential role in the cascade of events leading to egress of stem cells from the bone marrow to the peripheral blood. To investigate further the role of mature neutrophils and of reactive oxygen intermediates (ROIs), known to be involved in the signal transduction of neutrophils, we used mice deficient in respiratory burst, and thus the production of ROIs, to study the involvement of this activation pathway in stem cell mobilization. B6 mice with chronic granulomatous disease (CGD) received either cyclophosphamide (200 mg/kg) on day 1 and granulocyte colony-stimulating factor (G-CSF) (250 microg/kg/d) on days 3-6 or a single dose of interleukin 8 (IL-8; 30 microg/mouse) as a mobilization regimen. On day 7, the number of stem and progenitor cells in blood and bone marrow was compared with control B6 animals (with intact respiratory burst). White blood cell counts, bone marrow cellularity and the frequency of granulocyte-macrophage colony-forming cells (GM-CFC), and cobblestone area-forming cells (CAFC) on days 7 (CAFC-7) and 28 (CAFC-28) were determined. After cyclophosphamide and G-CSF (CY + G), both mouse strains showed considerable mobilization of CAFC-7 and CFU-GM to the blood. Normal mice showed up to a 1905-fold increase in progenitors per ml blood, whereas CGD mice showed up to a 264-fold increase in blood progenitors. IL-8 also induced mobilization in both mouse strains. In addition to progenitors, primitive stem cells measured as CAFC-28 and as CAFC at day 35 were also mobilized by both mobilization protocols in normal as well as in CGD mice. In conclusion, respiratory burst and the subsequent signal transduction pathway do not appear to be required for mobilization of stem cells. Accordingly, neutrophils either are not involved in stem cell mobilization or other signalling pathways within neutrophils must exist that lead to the release of factors which activate stem cell egress from the bone marrow.

G-CSF is routinely used to hasten recovery from chemotherapy-induced neutropenia. We have recently shown that G-CSF, when combined with stem cell-damaging cytotoxic agents, results in enhanced stem cell damage and loss of marrow reserve. To investigate the mechanisms of stem cell damage caused by G-CSF, we gave C57BL/6 (B6) mice repeated doses of cyclophosphamide ([CY] 84 mg/kg) or carmustine ([BCNU] 13.2 mg/kg) and G-CSF (250 microg/kg/day) for either four days or eight days. Two different regimens of G-CSF were chosen to study the influence of increased proliferation on hematopoiesis which was measured at the end of the first, third and sixth 14-day cycle of each cytotoxic agent and 7 and 20 weeks after completion of all cycles. A spectrum of hematopoietic indices was measured including WBC, bone marrow cellularity, granulocyte/macrophage-colony-forming cells (GM-CFC), colony-forming cells with high proliferative-potential (HPP-CFC), cobblestone area-forming cells ([CAFC]-day 7 and CAFC-day 28), and long-term marrow repopulating ability in vivo. Despite the absence of differences in peripheral blood cell counts or bone marrow cellularity 14 days after each dose, progenitor cell levels (HPP-CFC, GM-CFC, and CAFC-7) were increased up to 2.5-fold with cytotoxic agent and G-CSF administration compared with cytotoxic agent administration alone. Mice given G-CSF for eight days had the greatest number of progenitors suggesting a dose-response relationship for G-CSF administration. G-CSF resulted in a decrease in hematopoietic stem cell (CAFC-28) content when measured two weeks after each cycle of saline, CY, and BCNU. Twenty weeks after six cycles of BCNU, the reduction in stem cell levels persisted and was further decreased when G-CSF was added to BCNU for four or eight days. Data from this study suggest that the most likely explanation for the damaging effects of G-CSF is that G-CSF directly or indirectly induces stem cells to differentiate into more committed hematopoietic cells resulting in a loss of marrow reserve. This effect is enhanced in animals with an already compromised hematopoietic stem cell compartment as seen with repeated doses of BCNU.

The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1(b) mice received 30 x 10(6) GPI-1(a) marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S-phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity.

The Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of the S. pombe Rad17 (Rad17Sp) protein. The human RAD17Sp open reading frame (ORF) encodes a protein of 681 amino acids; the mRAD17Sp ORF codes for a protein of 688 amino acids. The mRAD17Sp messenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to the S.pombe Rad17Sp protein. Sequence homology was also noted with the Saccharomyces cerevisiae Rad24Sc (which is the structural counterpart of S.pombe Rad17Sp) and structurally related polypeptides from Caenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii, and Drosophila melanogaster. The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a "Walker A" motif. Using FISH and analysis of a panel of rodent-human cell hybrids, the human RAD17Sp gene (HGMW-approved symbol RAD17 could be localized on human chromosome 5q13-q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes., (Copyright 1999 Academic Press.)

Bone marrow stem cells collected from B6-Gpi-1a mice pretreated with 5-fluorouracil were incubated for 2 h at 37 degrees C in the presence of the recombinant adenovirus-associated virus-based vector (rAAV) SSV9. As measured in vitro immediately following transduction, SSV9 was found to be effective in transducing the primitive cobble-stone-area-forming cell (CAFC)-35 subset (60% transduction efficiency). However, this did not predict long-term expression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 106 transduced bone marrow stem cells into lethally irradiated recipients. CAFC analysis of bone marrow cells and Southern blot analysis of bone marrow and spleen cells were negative, and polymerase chain reaction analysis showed less than 0.1% transduction in bone marrow cells. Therefore, based on our study we conclude that rAAV transiently transduces hematopoietic stem cells but fails to integrate into the genome, leading to the loss of the reporter gene within the first six months after transplantation in vivo.

The Schizosaccharomyces pombe rad1+ cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human RAD1 (hRAD1) and mouse RAD1 (mRAD1) homologs of the S. pombe Rad1 (Rad1) protein. The human RAD1 open reading frame (ORF) encodes a protein of 282 amino acids; the mRAD1 ORF codes for a protein of 280 amino acids. The human RAD1 and mRAD1 messengers are highly expressed in the testis as different mRNA species (varying from 1.0, 1.4, 1.5, to 3.0 kb). The hRAD1 and mRAD1 proteins are 30% identical and 56% similar to the S. pombe Rad1 protein. Sequence homology was also noted with the Saccharomyces cerevisiae Rad17p, the putative 3'-5' exonuclease Rec1 from Ustilago maydis, and the structurally related polypeptides from Arabidopsis thaliana and Caenorhabditis elegans. The degree of conservation between the mammalian RAD1 proteins and those of the other species is consistent with the evolutionary distance between the species, implicating that these proteins are most likely true counterparts. Together, this suggests that the structure and function of the checkpoint "rad" genes in the G2/M checkpoint pathway are evolutionarily conserved between yeasts and higher eukaryotes. The human RAD1 gene could be localized on human chromosome 5p13, a region that has been implicated in the etiology of small cell lung carcinomas, squamous cell carcinomas, adenocarcinomas, and bladder cancer., (Copyright 1998 Academic Press.)

Despite the increasing use of cytokines to circumvent the acute dose-limiting myelotoxicity of cancer treatment, little is known about the combined effects of cytotoxic agents and cytokines on the primitive stem cells responsible for long-term hematopoiesis. In an experimental model, we administered cytotoxic agents that have variable effects on primitive stem cells in C57BL/6 (B6)-mice. Mice received six every-other-week doses of cyclophosphamide (CY, 84 mg/kg), VP-16 (24 mg/kg) + cisplatinum (2.4 mg/kg), carboplatinum (50 mg/kg), chlorambucil (12 mg/kg), BCNU (13.2 mg/kg), or TBI (80 cGy). Granulocyte colony-stimulating factor (G-CSF; 250 microg/kg/day) was administered subcutaneously twice daily on days 3 to 6 after each dose of the cytotoxic agent. Comparison with animals receiving the cytotoxic agent alone was made to investigate the effects of G-CSF on long-term hematopoiesis. Hematopoiesis was measured 20 weeks after the last dose of the cytotoxic agent by assessment of peripheral blood counts, marrow cellularity, progenitor cell content (colony-forming units-spleen; CFU-S), and primitive stem cell number (long-term repopulating ability and day 28 and day 35 cobblestone area-forming cell [CAFC] frequencies). Exposure to cytotoxic agents alone resulted in a significant decrease in primitive stem cells (as measured by repopulating units [RU] and day 28 and day 35 CAFC content) in animals given carboplatinum, chlorambucil, BCNU, and TBI, but not in animals treated with cyclophosphamide or VP-16 and cisplatinum. The addition of G-CSF resulted in a significant decrease in stem cell content when compared with no G-CSF administration in animals treated with chlorambucil, BCNU, or TBI. Thus, G-CSF administered after repeated exposure to cytotoxic agents, appeared to damage the primitive stem cell compartment when used in combination with agents known to damage primitive stem cells. These results, although obtained in an experimental model, should raise concerns for the indiscriminate use of G-CSF in the clinic., (Copyright 1998 by The American Society of Hematology.)

Transcription-coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair- and transcription-competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10-15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell-free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 microg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 10(7) congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 x 10(7) or 3 x 10(7) donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 +/- 2.7), compared with 5-FU alone (18.5 +/- 2.6, P < .0005), or saline controls (17.8 +/- 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 microg/dose) was added to a reduced dose of KL (12.5 microg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

Various cytokines have been reported to have radioprotective effects on the bone marrow. Of these, c-kit-ligand (KL) and interleukin-1 (IL-1) have the most dramatic effect when given prior to total body irradiation (TBI). Given simultaneously, KL and IL-1 demonstrated a strong effect on increasing the LD50/30 of mice. In this case the LD50/30 of C57BL/6 mice was 1.25 (1.14-1.38) times higher (10.08 Gy [confidence interval (c.i.): 9.62-10.56] vs. 8.05 Gy [c.i.: 7.64-8.42]) when KL (120 micrograms/kg) and IL-1 (40 micrograms/kg) were injected subcutaneously at 20 hours before TBI. It was also investigated whether the combined effects of KL and IL-1 resulted in changes in the intrinsic radiation sensitivity of different bone marrow subsets. Therefore, mice were irradiated and the survival of bone marrow subsets was determined at 4-6 hours after TBI by using the CFU-S assay and the competitive repopulation assay. The CFU-S subset displayed an increased Dzero value in KL and IL-1-treated mice (0.88 Gy vs. 0.72 Gy) and the protection factor was 1.22, close to the factor found for the hemopoietic syndrome (LD50/30). It may therefore be concluded that CFU-S are the target cell population involved in hemopoietic death. Additional protection of the more primitive stem cell subset with long-term repopulation ability (LTRA) could not be shown from the data we obtained with the competitive repopulation assay. Both Dzero and the extrapolation number (n) were increased, but not significantly. These data suggest that radioprotection by cytokines is caused mainly by the decreased radiation sensitivity of the CFU-S subset, although earlier subsets may also be protected (but to a lesser extent).

Fractionated and low-dose-rate total-body irradiation (TBI) were compared with single-dose high-dose-rate TBI for induction of long-term hemopoietic chimerism in a murine syngeneic bone marrow transplantation model. At 5 months after TBI and bone marrow transplantation, the degree of stable blood chimerism was determined from the proportion of stem cell-derived donor (B6-Gpi-1a) and host (B6-Gpi-1b) blood erythrocytes. This end point was used to construct radiation dose-response curves for long-term donor marrow engraftment corresponding to ablation of primitive bone marrow stem cells of the host. Increasing dose fractionation and decreasing dose rate had the effect of restoring host hemopoiesis and required higher TBI doses for equal donor engraftment. Most of the dose recovery occurred within the first 6 h between fractions, consistent with the kinetics of sublethal damage repair. The late chimerism data were fitted to the linear-quadratic model using indirect and direct analysis for a fixed threshold response. Both analyses gave relatively low alpha/beta ratios (below 2 Gy), within the range normally seen in late-responding tissues. The dose-rate data gave a repair half-time of 2 h as estimated by the incomplete-repair model. These estimates contrast with the much higher alpha/beta values and lower repair half-times derived from acute hemopoietic failure as indicated by LD50/30, with the implication that separate target cell populations with differing radiosensitivities are involved in these two bone marrow end points.

The omental lymphoid organ (OLO) is a part of the greater omentum composed of vascularized milky spots situated between fat cells and containing lymphocytes, plasma cells and macrophages. We analysed the disappearance of intraperitoneally injected tumor cells from the peritoneal cavity and their infiltration into and disappearance from the OLO and the parathymic lymph nodes (PTLN) that drain the peritoneal cavity. After intraperitoneal inoculation of irradiated syngeneic tumor cells, they were visible in the OLO within 24 h. After 3 days, no tumor cells were seen anymore, but there were many macrophages that had phagocytosed tumor cells. After intraperitoneal inoculation of nonirradiated syngeneic tumor cells, a solid growing tumor mass developed in the OLO. The PTLN were also invaded by these nonirradiated tumor cells within 24 h as transfer of cell suspensions of these lymph nodes into naive mice leads to the death of the recipient mice due to tumor growth. However, from day 6 onwards, after tumor inoculation, these lymph nodes contained no tumor cells, despite progressive tumor growth intraperitoneally and in the liver and lungs. In allogeneic mice, tumor cells were rejected in the OLO after 7-10 days. Simultaneously, the number of lymphocytes and macrophages increased and a plasma cell reaction developed. The PTLN did not have tumor-inducing potency at any stage after intraperitoneal tumor injection. This study suggests, that the PTLN are more effective in the (local) eradication of tumor cells than the OLO. Still, considering its immunological potential, the OLO might be important as 'inducer' of immunological reactions in the peritoneal cavity.