Sphingosine kinase 1 expression is downregulated during differentiation of Friend cells due to decreased c-MYB.

Sphingosine kinase 1 (SPHK1) overexpression in malignant cells has been reported. Mouse Friend cells showed higher SPHK1 but not SPHK2 expression compared with other mouse cell lines. A Sphk1 promoter analysis demonstrated the region between -53bp and the first exon as the minimal promoter. Further promoter truncation revealed the importance of a MYB-binding site. EMSA using this region as the probe demonstrated one band containing c-MYB protein, and its intensity decreased during erythroid differentiation with hexamethylane bisacetamide (HMBA), a potent inducer of erythroid differentiation of Friend cells. ChIP assay also revealed in vivo binding of c-MYB. c-MYB overexpression and siRNA for c-Myb affected SPHK1 expression, confirming the important regulatory role of c-MYB in SPHK1 expression. HMBA reduced c-MYB expression rapidly. Induced differentiation by HMBA caused a marked and rapid reduction of SPHK1 mRNA, protein and enzyme activity leading to the rapid decrease of cellular sphingosine 1-phosphate level. Moreover, terminally differentiated cells did not resume SPHK1 expression. Compared with original Friend cells, stable overexpression of wild-type SPHK1 showed higher cell proliferation, resistance to cell death by serum depletion. Interestingly, HMBA-induced differentiation of these cells was delayed but not completely suppressed. In contrast, SPHK inhibitor and its siRNA inhibited cell growth and enhanced HMBA-induced differentiation significantly, suggesting that SPHK1 delayed HMBA-induced differentiation by its cell proliferation-promoting activity. Effects of pertussis toxin, a G-protein-coupled receptor inhibitor, and S1P receptor antagonist on Friend cell growth and differentiation were negligible, suggesting the importance of the intracellular SPHK1/S1P signaling in Friend cells.

p27 deregulation by Skp2 overexpression induced by the JAK2V617 mutation.

Janus kinase 2 (JAK2) V617F mutation has been regarded as the major cause of myeloproliferative disorders (MPD). However, the mechanisms of abnormal cell growth by JAK2V617F have not been elucidated. In this study, cell cycle regulatory protein expression was analyzed using JAK2V617F-Ba/F3 and mock-Ba/F3. JAK2V617F-Ba/F3, but not mock-Ba/F3, showed IL-3 independent cell growth and constitutive STATs activation. Deregulation of p27(Kip1), the cell cycle regulator at the G1 to S transition, was observed in JAK2V617F-Ba/F3 but not in mock-control. p27(Kip1) deregulation was not due to p27(Kip1) mRNA level but due to high Skp2 expression, a subunit of ubiquitin E3 ligase, through the STAT binding in the Skp2 promoter. Like JAK2V617F overexpression, constitutively active STAT5 or STAT3 induced aberrant p27(Kip1) expression of Ba/F3 cells. Similar findings were observed in BCR/ABL-transfected Ba/F3. Our results elucidate the regulatory mechanism by which JAK2V617F modulates Skp2 gene expression through the STAT transcription factors.

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5' promoter.

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T-->G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.

Skewed X chromosome inactivation in fraternal female twins results in moderately severe and mild haemophilia B.

Female carriers of haemophilia B are usually asymptomatic; however, the disease resulting from different pathophysiological mechanisms has rarely been documented in females. In this study, we investigated the mechanisms responsible for haemophilia B in fraternal female twins. We sequenced the factor IX gene (F9) of the propositus, her father, a severe haemophilia B patient and the other family members. X chromosome inactivation was assessed by the methylation-sensitive HpaII-PCR assay using X-linked polymorphisms in human phosphoglycerate kinase 1 gene (PGK1) and glutamate receptor ionotropic AMPA 3 gene (GRIA3). The twins were found to be heterozygotes with a nonsense mutation (p.Arg384X) inherited from their father. The propositus, more severely affected twin, exhibited a significantly higher percentage of inactivation in the maternally derived X chromosome carrying a normal F9. The other twin also showed a skewed maternal X inactivation, resulting in a patient with mild haemophilia B. Thus, the degree of skewing of maternal X inactivation is closely correlated with the coagulation parameters and the clinical phenotypes of the twins. Furthermore, we identified a crossing-over in the Xq25-26 region of the maternal X chromosome of the more severely affected twin. This crossing-over was absent in the other twin, consistent with their fraternal state. Differently skewed X inactivation in the fraternal female twins might cause moderately severe and mild haemophilia B phenotypes, respectively.

Implications of sphingosine kinase 1 expression level for the cellular sphingolipid rheostat: relevance as a marker for daunorubicin sensitivity of leukemia cells.

We recently reported increased sphingosine kinase 1 (SPHK1) and decreased neutral sphingomyelinase 2 (NSMase2) gene expression in myelodysplastic syndromes and acute leukemia. This alteration is supposed to change the cellular sphingolipid metabolites; however, positive correlations were observed between daunorubicin (DA)-IC50 and the SPHK1 message but not between DA-IC50 and NSMase2 messages, when 16 different leukemia cell lines were used to analyze the relationship between gene expressions and chemosensitivity against DA. Using two cell lines with either the highest or lowest SPHK1 expression, cellular ceramides and sphingosine 1-phosphate (S1P) were quantified by liquid chromatography/mass spectrometry. Increased ceramide was observed in DA-sensitive, but not in DA-resistant cell lines treated with low doses of DA. Upon DA treatment, S1P decreased more in the sensitive cell lines than in resistant cell lines. A SPHK inhibitor recovered the DA sensitivity of DA-resistant cells. The modulation of SPHK1 gene expression by either overexpression or using siRNA affected the DA sensitivity of representative cell lines. Results clearly show that SPHK1 is both a good marker to predict the DA sensitivity of leukemia cells and a potential therapeutic target for leukemia with high SPHK1 expression, and suggest that the sphingolipid rheostat plays a significant role in DA-induced cytotoxicity.

In vitro characterization of missense mutations associated with quantitative protein S deficiency.

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T-->C transitions: implication of wobble base pair formation at the nucleotide incorporation step.

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T-->C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T-->C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

Mutational analysis of the PTEN/MMAC1 gene in non-Hodgkin's lymphoma.

The PTEN/MMAC1 gene at 10q23.3, which has dual specific phosphatase activity, is a novel tumor suppressor gene candidate. Various kinds of tumors have mutations in this gene, including glioblastoma, endometrial carcinoma and prostate cancer. We examined 29 cases of primary non-Hodgkin's lymphoma (NHL) for mutations in the PTEN/MMAC1 gene. One case of diffuse large B cell lymphoma had an 11 bp deletion, but the remaining 28 cases showed no mutations in the genome. Two of these 28 cases showed missense mutations in the PTEN/MMAC1 transcripts, but no alterations in the genomic DNA. These mRNA missense variants are similar to PTEN/MMAC1 transcript aberrations which have been reported in patients with breast cancer. These findings suggest that alterations in the PTEN/MMAC1 gene play a role in the pathogenesis of NHL.

Growth analysis of marrow CD34-positive hematopoietic progenitor cells in patients with myelodysplastic syndromes.

The in vitro colony-forming capacities of marrow CD34-positive (CD34+) cells from 25 patients with myelodysplastic syndromes (MDS) were studied. In all patients this purified cell population showed erythroid (burst-forming unit erythroid; BFU-E) or non-erythroid colony growth, both of which were more restricted than non-purified mononuclear cell population under stimulation with erythropoietin (EPO) or granulocyte/macrophage colony-stimulating factor (GM-CSF) alone. MDS patients examined were put into two groups; refractory anemia (RA) or RA with ringed sideroblasts (RA/RARS) and RA with excess of blasts (RAEB) or RAEB in transformation (RAEB/RAEB-t). The CD34+ cells of both RA/RARS and RAEB/RAEB-t exhibited a similarity to the cells of normal individuals in their responsiveness to the addition of interleukin 6 (IL-6), IL-3 or stem cell factor (SCF). SCF caused powerful but highly variable responses in both erythroid and nonerythroid colony formation as compared with IL-6 or IL-3. We demonstrated that MDS marrow hematopoietic progenitor cells reactive to GM-CSF or EPO were additionally stimulated with early-acting hematopoietic growth factors including IL-6, IL-3 and SCF in a fashion similar to those of normal individuals.

Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP).

The human androgen-receptor gene (HUMARA) has been used for analysis of X chromosome inactivation (XCI) pattern because of a polymorphic short tandem repeat (STR) near the 5'-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzes methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. Because DNA modified by sodium bisulfite is also available for assessment of methylation status of other genes by setting specific primers for them, we performed the simultaneous assessment of clonality and aberrant hypermethylation of p15INK4B gene in myelodysplastic syndromes. These simultaneous assessments were easily possible and provided much information despite requiring only a small volume of DNA. The HUMARA-MSP assay may facilitate the analyses for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability. Leukemia (2000) 14, 207-212.

Clonality analysis of refractory anemia with ring sideroblasts: simultaneous study of clonality and cytochemistry of bone marrow progenitors.

X chromosome inactivation and polymorphism of the human androgen receptor (HUMARA) gene has been applied for analyzing the clonality of blood cells. In the present study, the clonal relationship was investigated between peripheral blood polymorphonuclear cells (PMNCs) and marrow progenitor cells and the origin of ringed sideroblasts in patients with refractory anemia with ring sideroblasts (RARS) by polymerase chain reaction (PCR) of HUMARA gene. The X-inactivation patterns of circulating PMNCs and T lymphocytes as well as individual granulocyte colonies grown in vitro from bone marrow cells were analyzed. The development of ringed sideroblasts in erythroid colonies by iron staining and their X-inactivation pattern were also examined. All three RARS patients showed monoclonal PMNCs. In granulocyte colonies, however, two different X-inactivation patterns were observed in all patients, indicating that non-clonal progenitor cells remained in the bone marrow. All erythroid colonies consisted of ringed sideroblasts exclusively showed one pattern dominant in those of PMNCs. Our findings suggest that non-clonal progenitor cells persist in some RARS cases, that erythroid progenitors show mosaicism, and that ringed sideroblasts may be derived from an abnormal clone involved in the pathogenesis of this disease.

Establishment and characterization of a novel cell line, TK-6, derived from T cell blast crisis of chronic myelogenous leukemia, with the secretion of parathyroid hormone-related protein.

We established a novel T cell line, designated TK-6, from a patient with T cell lineage blast crisis of chronic myelogenous leukemia (CML) complicated by hypercalcemia. A surface marker study showed T cell phenotype, cluster designation (CD)4, CD5 and CD7. Light and electron microscopic examination revealed myeloperoxidase (MPO)-negative, however, ultrastructural examination under certain specific conditions demonstrated that some cells were MPO-positive. The TK-6 cell karyotype carried a t(9;22)(q34;q11) and additional chromosome aberrations, including a deletion of the long arm of chromosome 6 and the abnormality of chromosome 7. Southern blot analysis showed rearrangement of the T cell receptor beta-chain (TCR beta) gene and the major breakpoint cluster region (bcr) gene. Northern blot analysis detected the expression of the parathyroid hormone-related protein (PTHrP) gene, however, the proviral genome of human T cell leukemia virus type I (HTLV-I) was negative. This cell line will provide a valuable resource for the analysis of the relationship between T cell lineage crisis and myeloid differentiation and for the analysis of humoral hypercalcemia of malignancy (HHM) or leukemia.

Truncated c-Myb expression in the human leukemia cell line TK-6.

The c-MYB proto-oncogene encodes a transcription factor which plays an important role in hematopoiesis. We detected truncated c-MYB mRNA (2.0 kb) and c-Myb protein (55 kDa) in the TK-6 cell line, which was established from a patient with chronic myelogenous leukemia in T cell blast crisis. Mutated c-MYB cDNA clone (WTK-1) was isolated from a TK-6 cell cDNA library and sequenced in its entirety. Compared with the wild-type human c-MYB sequence, the WTK-1 sequence diverged at the 3' ends of exons 9. A termination codon was present as the second codon downstream from the point of divergence and was followed by a previously unknown rearranged sequence. The conceptual protein encoded by WTK-1 (Myb(TK-6)) comprises 402 amino acids and lacks the negative regulatory domain of the normal c-Myb, reminiscent of the activated form of Myb protein. Luciferase reporter assay in NIH3T3 cells showed that the expression vector encoding Myb(TK-6) stimulated Myb-regulated mim-1 promoter more effectively than that encoding wild-type human c-Myb, suggesting that Myb(TK-6) is functional as a transcription factor, and thus as a potential transforming protein. Southern blot and mutant allele-specific polymerase chain reaction analyses showed that the same rearrangement of the c-MYB gene in TK-6 was present in late, but not in early, specimens obtained from the patient, indicating that this mutation had been acquired during disease progression.

Cytogenetic and clonal culture evaluation after response to low dose Ara-C in myelodysplastic syndromes.

Cytogenetic and bone marrow culture studies were performed sequentially in 13 patients with myelodysplastic syndromes (MDS) who responded to low dose cytosine arabinoside (Ara-C) treatment (complete in nine and partial in four patients). Of nine patients with initial clonal karyotypic abnormalities, six recovered a normal karyotype after attaining a response to treatment, but the other three patients retained partial or total karyotypic abnormalities. A new clonal karyotypic abnormality appeared after treatment in one patient. Eight patients showed normal colony growth of both granulocyte-macrophage colony-forming units and erythroid burst-forming units after treatment, but five were still defective. There was a clear difference in the duration of response to treatment between these two groups. Consolidation treatment was not effective in patients with persistent karyotypic abnormalities or defective colony formation. Although the number of patients studied is small, these results suggest that hemopoiesis in patients with MDS following a response to treatment with low dose Ara-C is heterogeneous. Consolidation chemotherapy is recommended to ensure and prolong the response in patients showing normalization of both cytogenetic and bone marrow culture results.

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes.

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4B methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Establishment and characterization of an immature human megakaryoblastic cell line, MEG-A2.

We have established a novel human megakaryoblastic cell line, designated as MEG-A2, from a patient with megakaryoblastic crisis of Philadelphia (Ph) chromosome positive chronic myelogenous leukemia. MEG-A2 cells showed positive phenotypes for periodic acid Schiff and alpha-naphthylbutyrate esterase reactions, but were negative for myeloperoxidase and naphthol ASD chloroacetate esterase reactions. Flow cytometric analyses of cell surface markers revealed that MEG-A2 cells had a low level of GP IIb/IIIa expression as well as apparent expressions of CD4, CD7, CD13, CD33 and CD34 antigens, but no expression of GP Ib nor glycophorin A. Stimulation with phorbol 12-myristate 13-acetate (PMA) dramatically increased the expression of megakaryocyte-related markers such as HPL-3, J15, Pit-1, Y2/51 and AN51 in MEG-A2 cells. The PMA-stimulation also induced expression of platelet peroxidase (PPO) in MEG-A2 cells on electromicroscopic observation. Proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or erythropoietin were observed, and the expression of GP IIb/IIIa was increased by stimulation with GM-CSF, IL-3, erythropoietin and interleukin-6 (IL-6). Protein S mRNA expression was seen in cultured cells on Northern blot analysis. Expression of platelet factor 4 mRNA was induced in PMA-stimulated cells, and a marked accumulation of protein was observed in the culture medium. In conclusion, a new cell line, MEG-A2, belongs to the relatively immature megakaryocytic lineage and has markedly increased megakaryocytic characteristics with PMA stimulation.

Histone deacetylase inhibitors are the potent inducer/enhancer of differentiation in acute myeloid leukemia: a new approach to anti-leukemia therapy.

We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, beta-globin) were silent or downregulated, and housekeeping genes (beta-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.

Maturation sequence hierarchy of murine macrophage progenitor cells.

The heterogeneity of murine macrophage progenitors was analyzed with special interest in their maturation sequence. The gross appearance of macrophage colonies formed by C57BL/6 marrow cells was classified into three types: compact, intermediate, and dispersed. Progenitors of each type could be segregated by sedimentation velocity. Findings of cell-cycle studies showed that different types of colonies were not derived from a single progenitor cell at different times in the cell cycle. Therefore, it is considered that the difference in colony types can be attributed to the heterogeneity of progenitors. Suspension cultures of fractionated marrow cells rich in compact, intermediate, and dispersed colonies yielded mainly intermediate colonies, dispersed colonies, and clusters, respectively. As suspension cultures containing colony stimulating factor were considered to induce cell division as well as maturation, it is supposed that progenitors of compact, intermediate, and dispersed colonies constitute a maturation sequence hierarchy in this order.

Age-related changes in the function of hemopoietic stroma in mice.

Hemopoietic function of the marrow and splenic stroma in C57BL/6 mice aging from 2 to 24 months were evaluated with the use of subcutaneous implantation method. Although there was no significant difference in the concentration as well as the total number of CFU(S) in femoral marrows between aged and young mice, the rates of CFU(S) repopulation in the femoral marrows from aged donors were significantly lower than in those from young donors when grafted subcutaneously in the 2 month-old syngeneic hosts. On the other hand, the femoral marrow grafts from 2 month-old donors were constantly repopulated irrespective of the age of the hosts. An essentially similar finding was obtained in the study of spleen grafts. It is suggested that the defective hemopoiesis, if any, in aged mice is primarily caused by the changes in hemopoietic stroma rather than those in hemopoietic stem cells.