Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, inhibits the growth of squamous cell carcinoma (SCC) cells in vitro and in vivo by down-regulating expression of heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) and FGF-2.

Heparin-binding protein 17 (HBp17)/fibroblast growth factor-binding protein-1 (FGFBP-1) was first purified from medium conditioned by A431 cells for its capacity to bind to fibroblast growth factors 1 and 2 (FGF-1 and -2). Among FGF family members, FGF-2 is a potent mitogen for various cell types, including vascular endothelial cells, fibroblasts, and cancer cells such as oral squamous cell carcinoma (OSCC) cells. Besides being well known in bone metabolism, the active form of vitamin D3, i.e., 1α,25(OH)2D3 (1,25D3), was reported to have protective effects for heart disease and cancer. Previously, we reported that 1,25D3 inhibited HBp17/FGFBP-1 expression in OSCC cell lines through NF-κB inhibition (IκBα activation) and resulted in the inactivation of FGF-2. In this study, we examined the potential anti-tumor effect of ED-71, an analog of 1α,25(OH)2D3, for squamous cell carcinoma cells in vitro and in vivo. The cell lines used were OSCC cell lines (NA-HO-1-n-1 and UE-HO-1-u-1), established from oral cancer patients in our laboratory, and an epidermoid carcinoma/SCC cell line (A431). The growth assay in serum-free culture revealed that ED-71 inhibited the growth of the cancer cell lines in a dose-dependent manner. In addition, ED-71 suppressed HBp17/FGFBP-1 expression by inhibiting the NF-κB pathway as did 1,25D3. Furthermore, a luciferase reporter assay revealed that the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) was down-regulated by ED-71. Oral administration of ED-71 significantly inhibited the growth of A431-derived tumors in athymic nude mice. Immunohistochemical analysis revealed that the expression of HBp17/FGFBP-1, FGF-2, CD31, and Ki-67 in the tumors of ED71-treated group was down-regulated in comparison to control. These results suggest that ED-71 possesses potential anti-tumor activity for SCCs both in vitro and in vivo. This compound may act directly on the tumor cells or on endothelial cells by modulating the tumor microenvironment.

Eldecalcitol (ED-71), an analog of 1α,25-dihydroxyvitamin D3 as a potential anti-cancer agent for oral squamous cell carcinomas.

We have previously reported that 1,25(OH)2D3 inhibits NF-κB activity and thus inhibits growth of OSCC cells in serum-free culture and down-regulates HBp17/FGFBP-1 expression, which is important for cancer cell growth and angiogenesis. Here, we have investigated the effects of ED-71, an analog of vitamin D3 (VD) on OSCC cell lines in serum-free culture. It is known that ED-71 has a stronger inhibitory effect on bone resorption compared to VD and other VD analogs. To the best of our knowledge, there was no report examining the potential of ED-71 as an anti-cancer agent for OSCC. We found that ED-71 is able to inhibit the growth of cancer cell lines at a concentration of hundred times lower than calcitriol. As Cyp24A1 was reportedly induced in cancer cells, we measured the expression of CYP24A1 in OSCC cell lines (NA and UE), A431 epidermoid carcinoma and normal fibroblast cell (gfi) in serum-free culture. As a result, CYP24A1 mRNA and the protein expression in the OSCC cells treated with ED-71 increased in a dose-dependent manner. However, in vivo experiment, in which the A431 cells were implanted in mice, tumor formation was reduced by the ED-71 treatment with no significant difference between Cyp24A1 expression in the tumors of ED-71-treated and control group, as analyzed by western blotting and immunohistochemistry. These results suggest that ED-71 is a potential anti-cancer agent for OSCC.

1α,25OH2D3 down-regulates HBp17/FGFBP-1 expression via NF-κB pathway.

The heparin binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1, GenBank accession no. NP-005121) has been reported to enhance angiogenesis as well as promotes tumor growth in vivo. Furthermore, this molecule was found to be highly expressed in the tissue and cell lines of oral squamous cell carcinoma (OSCC). 1α,25(OH)2D3 is used to study its potential to curb the expression of HBp17/FGFBP-1 in cancer cells. Consequently, we found that HBp17/FGFBP-1 mRNA and protein levels were significantly down-regulated. In this present study, we show that this event takes place via the NF-κB pathway since mRNA and protein levels of this pathway regulator, IκBα, were found to be significantly up-regulated. Furthermore, the promoter activity of HBp17/FGFBP-1 (region between -217 and +61) measured by a luciferase reporter assay was down-regulated following treatment. Silencing of VDR with siRNA showed the effect of 1α,25(OH)2D3 on HBp17/FGFBP-1. Based on these findings, we concluded that 1α,25(OH)2D3 down-regulated HBp17/FGFBP-1 expression via NF-κB. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Infectious complications in chronic graft-versus-host disease: a retrospective study of 145 recipients of allogeneic hematopoietic stem cell transplantation with reduced- and conventional-intensity conditioning regimens.

To assess infectious complications associated with chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) with reduced- and conventional-intensity conditioning regimens (RIC, n=91; CIC, n=54, respectively), we retrospectively analyzed data from 145 consecutive patients with cGVHD after allogeneic HSCT from a human leukocyte antigen-matched related or unrelated donor. In the present retrospective analysis, 57% (83/145) of patients with cGVHD developed infections, with a mortality rate of 27% (22/83). The incidences of bacteremia (n=28), central venous catheter-related infections (n=11), bacterial pneumonia (n=4), invasive aspergillosis (n=7), and adenoviral hemorrhagic cystitis (n=8) were significantly higher in patients with prednisolone dose >or=1 mg/kg at the time of diagnosis of cGVHD. The present results suggest that infections associated with cGVHD, especially after high-dose prednisolone, are predictive of poor outcome regardless of whether the patient received RIC or CIC.

Characteristics of t(8;21) acute myeloid leukemia (AML) with additional chromosomal abnormality: concomitant trisomy 4 may constitute a distinctive subtype of t(8;21) AML.

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.

Non-DNA-binding Ikaros isoform gene expressed in adult B-precursor acute lymphoblastic leukemia.

Ikaros, a zinc finger transcription factor, is essential for lymphoid development. Mutant mice expressing dominant-negative Ikaros gene (Ikaros) isoforms develop an aggressive form of lymphoid malignancies. We examined the expression of Ikaros isoforms in 11 leukemic cell lines and adult acute lymphoblastic leukemia cells from 36 patients with B-precursor acute lymphoblastic leukemia (pre-B ALL) and nine with T-precursor acute lymphoblastic leukemia (pre-T ALL), using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In one pre-B ALL cell line, INC cells, and primary leukemic cells from 16 patients with pre-B ALL, we found the predominant expression of a non-DNA-binding Ikaros isoform, Ik-6. However, Ik-6 was not detected in pre-T ALL cells. All of pre-B ALL cells expressing Ik-6 were CD10(+), whereas CD10(-) pre-B ALL cells did not express Ik-6. The expression of Ik-6 was not related to karyotype abnormalities such as t(9;22) and t(4;11). Proteins from the cells that expressed Ik-6 alone failed to bind to the Ikaros protein-specific binding sequence in DNA. Ikaros proteins lacking the DNA binding sequences were detected in the cytoplasm but not in the nucleus of the cells. When INC and primary pre-B ALL cells that express Ik-6 alone were irradiated and cultured in the absence of serum, these cells produced functional Ikaros isoforms, Ik-1 and Ik-2. Purified CD19(+) CD10(-) and CD19(+) CD10(+) cells from normal human bone marrow did not express Ik-6. The predominant expression of Ik-6, which is the result of post-transcription dysregulation, is characteristic of adult pre-B ALL, especially CD10(+) pre-B ALL.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Aggressive neoplastic plasma cell growth with MLL gene rearrangement after high-dose therapy with autologous stem cell support for multiple myeloma.

We report a case of a patient with IgA kappa multiple myeloma (MM) mobilized with etoposide and subsequently receiving high-dose melphalan (HDM) with stem cell support. She relapsed rapidly post transplantation. Southern blot and fluorescent in situ hybridization analysis showed MLL gene rearrangement in the myeloma cells, which was not detected in the sample at diagnosis or in the PBSC harvested with etoposide plus G-CSF. These observations suggest that clonal rearrangement of the MLL gene is caused by etoposide. Patients with MM undergoing HDM with stem cell rescue may be at an increased risk of not only secondary leukemia, but also secondary genetic abnormalities in myeloma cells, especially those receiving priming with etoposide for peripheral blood stem cell collection.

Successful treatment with low-dose splenic irradiation for massive splenomegaly in an elderly patient with hairy-cell leukemia.

The therapeutic approach to hairy-cell leukemia (HCL) is in some instances still debated. Although management with alpha-interferon and purine analogues is well established, there is an alternative role for therapeutic splenectomy in patients with massive splenomegaly who have failed to respond to systemic therapy. Most patients with HCL will not be suitable for treatment with splenectomy as their ages at diagnosis are high. Here, we report an elderly Japanese HCL patient whose refractory massive splenomegaly responded well to low-dose splenic irradiation.

Expression of B cell-associated transcription factors in B-cell precursor acute lymphoblastic leukemia cells: association with PU.1 expression, phenotype, and immunogenotype.

B-lymphocyte development progresses through discrete stages characterized by regular DNA rearrangements of the immunoglobulin (Ig) loci that lead to the transcription of Ig genes and expression of B-cell antigen receptors. These developmental processes can also be distinguished by the expression of specific cell-surface markers. Therefore, rearrangement of the Ig, T-cell receptor (TCR) genes, and surface markers are generally considered as useful markers of the B- and T-cell lineage in lymphoproliferative disorders. However, concomitant rearrangement of Ig and TCR genes (double genotype) has been reported in the most immature lymphoid malignancies (lineage promiscuity), mainly in B-cell precursor acute lymphoblastic leukemia (pre-B ALL), but the mechanism is not fully understood. DNA rearrangements and specific cell-surface markers are regulated by several specific transcription factors. To better characterize the lineage promiscuity, we studied the relationship among the expression of lymphoid-associated transcription factors, phenotype, and immunogenotype. Rearrangement of the Ig light chain kappa gene was found in 37% of pre-B ALL samples and in all B-cell chronic lymphocytic leukemia (B-CLL) samples. Rearrangement of TCR gamma gene was shown in 40% of pre-B ALL samples but was not detected in any of the B-CLL samples. Among the tested B cell-associated transcription factors, Pax5 and E47 genes were expressed in all pre-B ALL and B-CLL samples. RAG-1 gene was expressed in all pre-B ALL samples but not in the B-CLL samples. Oct-2 gene was expressed in 82% of pre-B ALL and all B-CLL samples. The expression of PU.1 gene was shown in 56% of pre-B ALL but not in the B-CLL samples. Interestingly, the samples of pre-B ALL, which did not express the PU.1 gene, showed a significantly high frequency of TCR gamma gene rearrangement. This phenomenon was not found with Oct-2 gene expression. These findings suggest that absence of PU.1 expression may result in lineage promiscuity, such as the simultaneous rearrangements of Ig and TCR genes in pre-B ALL cells.

Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes.

A concanavalin-A-binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N-terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour-associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse-transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.

Rat hepatic 3 alpha-hydroxysteroid dehydrogenase: expression of cDNA and physiological function in bile acid biosynthetic pathway.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) [EC 1.1.1.213]2 plays important multifunctional roles in metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins and also in transforming the steroid nucleus for the biosynthesis of bile acids from cholesterol in liver. To gain insight into the details and physiological functions of 3 alpha-HSD in the bile acid biosynthetic pathway, cDNA clones of 3 alpha-HSD were isolated from rat liver lambda phage cDNA libraries by using specific antibodies to 3 alpha-HSD purified from rat liver. Transfection of the 3 alpha-HSD cDNA in Simian COS7 cells resulted in the expression of an immunoreactive protein to the antibodies against the purified enzyme, and the transfected cells exhibited activities for not only 7 alpha-hydroxy-5 beta-cholestan-3-one, the intermediate of bile acid biosynthesis, but also steroid hormones and 9,10-phenanthrenequinone. Northern blot analysis on poly(A)+ RNA by selective use of different cDNA fragments of the 5'-untranslated region, the coding region, and the 3'-untranslated region as probes revealed three hybridizable bands, 3.6, 2.7, and 2.5 kb, in liver and four bands, 3.6, 2.7, 2.5, and 1.8 kb, in ovary. Of these, the 2.7- and 1.8-kb bands were predominant in liver and ovary, respectively. Northern hybridization analysis also revealed that the coding region of the various sizes of mRNA seemed to be common. Southern blot analysis of genomic DNA by the selective use of the cDNA fragments as probes indicated that the various mRNA species were derived from a single gene, probably due to an alternative splicing mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae.

The cDNA coding for the precursor protein of rat liver mitochondrial vitamin D3 25-hydroxylase, cytochrome P450LMT25, was expressed under the control of the yeast alcohol dehydrogenase I promoter and terminator in Saccharomyces cerevisiae AH22 cells. The transformed yeast cells produced a P450LMT25 protein with an almost similar apparent molecular weight as compared with that of the authentic mature enzyme. The expression level of the P450LMT25 hemoprotein was about 5 x 10(4) molecules per cell as determined by reduced CO-difference spectra. The mitochondrial fraction prepared from the transformed yeast cells exhibited both 25-hydroxylase activity toward 1 alpha-hydroxyvitamin D3 and 27-hydroxylase activity toward 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in a reconstituted system containing bovine adrenodoxin and NADPH-adrenodoxin reductase.

Multi-functional property of rat liver mitochondrial cytochrome P-450.

To solve the problem of whether a common enzyme catalyzes both 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1 alpha-hydroxyvitamin D3 25-hydroxylation activity was 1.4 min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha-triol competitively inhibited 25-hydroxylation of 1 alpha-hydroxy-vitamin D3 and vice versa. From these results it was concluded that 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation and 1 alpha-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.

Unique property of liver mitochondrial P450 to catalyze the two physiologically important reactions involved in both cholesterol catabolism and vitamin D activation.

The cDNA for vitamin D 25-hydroxylase in rat liver mitochondria was transfected in COS cells in order to confirm our previous postulation that both 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol 27-hydroxylation and vitamin D 25-hydroxylation are catalyzed by a common enzyme. As a result it was found that both enzyme activities could be reconstituted from the solubilized extract of mitochondria of these cells, NADPH, NADPH-adrenodoxin reductase and adrenodoxin, giving unequivocal evidence that the two enzyme activities are catalyzed by a common enzyme.