Marrow stromal cells induce B7-H1 expression on myeloma cells, generating aggressive characteristics in multiple myeloma.

Tumor-associated B7-H1 molecules inhibit antitumor immunity in some malignancies. We found that B7-H1 expression on patient myeloma cells and human myeloma cell lines (HMCLs) was upregulated by cultivating the cells with autologous stromal cells and the human stromal cell line HS-5. Among major cytokines produced by HS-5 cells, interleukin (IL)-6-induced B7-H1 expression on HMCLs. Moreover, HS-5 cell-mediated B7-H1 expression was downregulated by inhibiting IL-6. B7-H1(+) HMCLs were more proliferative and less susceptible to antimyeloma chemotherapy compared with B7-H1(-) HMCLs. Moreover, the former cells showed higher levels of Bcl-2 and FasL expression than the latter. Finally, B7-H1 molecules on HMCLs induced T-cell apoptosis and anergy of tumor-specific T cells. Consistent with these in vitro observations, patients whose myeloma cells expressed high levels of B7-H1 had higher myeloma cell percentages in the bone marrow (BM) and higher serum lactate dehydrogenase levels compared with other myeloma patients. In addition, B7-H1 expression levels were often upregulated after myeloma patients relapsed or became refractory to therapy. Our data indicate that the BM microenvironment upregulates B7-H1 expression on myeloma cells, which links to the two biological actions of inducing T-cell downregulation and enhancing aggressive myeloma-cell characteristics. Modulating the B7-H1 pathway may be worthwhile in myeloma.

BRCA1 and FOXA1 proteins coregulate the expression of the cell cycle-dependent kinase inhibitor p27(Kip1).

We have previously shown that the breast cancer susceptibility gene, BRCA1, can transcriptionally activate the p27(Kip1) promoter. The BRCA1-responsive element was defined as a 35 bp region from position -545 to -511. We next determined that within this region is also a potential binding site for the transcription factor Forkhead box (FOX)A1. RNA and protein analysis as well as immunohistochemistry showed that expression of FOXA1 correlated with the expression of the estrogen receptor in a panel of breast cancer cell lines and tissues. In transient transfection reporter assays, FOXA1 could activate the p27(Kip1) promoter. Cotransfection of BRCA1 and FOXA1 resulted in a synergistic activation of the p27(Kip1) promoter. Mutation of the FOXA1 DNA-binding site in the p27(Kip1) promoter-luciferase construct significantly diminished the activity of FOXA1 alone or in combination with BRCA1. Cotransfection of FOXA1 and BRCA1 resulted in a greater amount of each protein compared to transfection of each expression vector alone. The half-life of FOXA1 was increased when coexpressed with BRCA1. Electrophoretic mobility shift assay analysis demonstrated that FOXA1 could bind to a wild-type oligonucleotide containing the FOXA1 binding site in the p27(Kip1) promoter, but this binding was lost upon mutation of this FOXA1 binding site. The protein-DNA binding complex could be supershifted with an antibody directed against FOXA1. The activity of the p27(Kip1) promoter as well as FOXA1 expression was reduced in cells treated with BRCA1 siRNA, thus silencing the expression of BRCA1 protein. In summary, we identified a FOXA1 binding site within the BRCA1-responsive element of the p27(Kip1) promoter and showed that FOXA1 activated the promoter alone and in conjunction with BRCA1. Furthermore, we identified high expression of FOXA1 in breast cancer cell lines and tissues, discovered a role for BRCA1 in the regulation of p27(Kip1) transcription and a possible interaction with BRCA1.

Near-triploidy and near-tetraploidy in hematological malignancies and mutation of the p53 gene.

Hyperdiploidy of > or =58 chromosomes is reported in 0.5-3% of hematological malignancies, but reports of near-triploidy (58-80 chromosomes) and near-tetraploidy (81-103 chromosomes), are few. We examined these chromosome abnormalities and analyzed the relationship with the mutation of the p53 gene. Thirty-one of 979 adult patients (3.2%) with hematological malignancies were identified as having near-triploid or near-tetraploid (tri-/tetraploid) chromosomes. These included 11 with B-cell neoplasms, seven with Hodgkin's lymphoma, five with T-cell neoplasms, four with myelodysplastic syndromes and four with acute myeloid leukemias. All patients had concurrent complex chromosome aberrations. Deletion of one allele of the p53 gene was found in two patients and a point mutation of the p53 gene was detected in five patients. Although abnormalities of the p53 gene have been reported in about 10% of hematological malignancies, these were found in seven of 31 (23%) patients with tri-/tetraploidy. These findings suggest that the abnormality of the p53 gene may be closely related with tri-/tetraploidy. The four myelodysplastic syndrome (MDS) patients with tri-/tetraploidy had a significantly worse prognosis than those with diploid cytogenetics (n = 35; P < 0.002). In B-cell neoplasms (n = 3), triploidy was associated with a worse prognosis than tetraploidy (n = 8) and diploidy (n = 130; P < 0.02).

Initial medical management of patients severely irradiated in the Tokai-mura criticality accident.

A nuclear criticality accident occurred in Japan on September 30, 1999, which resulted in severe exposure of three victims to mixed flux of neutrons and gamma-rays. Estimated average doses for the three victims were 5.4 Gy of neutrons and 8.5 Gy of gamma-rays for Patient A, 2.9 Gy of neutrons and 4.5 Gy of gamma-rays for Patient B, and 0.81 Gy of neutrons and 1.3 Gy of gamma-rays for Patient C. They then suffered the consequences of the effects of ionizing radiation resulting in acute radiation syndrome. In Patients A and B, bone marrow failure was so severe that they received haematopoietic stem cell transplantation. The graft initially took successfully in both patients, although in Patient B it was later taken over by his own haematopoietic cells. They also suffered from severe skin lesions, later exhibited gastrointestinal bleeding and eventually died of multiple organ failure 82 and 210 days after the accident, respectively. The survival of these patients beyond the period of agranulocytosis means that bone marrow failure per se caused by exposure to ionizing radiation may now be overcome. Patient C also developed bone marrow failure and was treated with granulocyte colony-stimulating factor as well as supportive care. He recovered without major complications and is now under periodical follow-up. Remarkably, during the prodromal phase, all the patients exhibited hypoxaemia, two of whom also showed interstitial oedema of the lungs. In Patient C these manifestations improved within a week. The circumstances of the accident and the initial medical treatment of the victims are described.

Abnormality of c-kit oncoprotein in certain patients with chronic myelogenous leukemia--potential clinical significance.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome and bcr/abl gene rearrangement which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate the biological properties of KIT in CML leukemogenesis, we performed analysis of alterations of the c-kit gene and functional analysis of altered KIT proteins. Gene alterations in the c-kit juxtamembrane domain of 80 CML cases were analyzed by reverse transcriptase and polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT --> AAG, Asn --> Lys), and six cases had the same base abnormality at codon 541 (ATG --> CTG, Met --> Leu) in the juxtamembrane domain. Because the change from Met to Leu at codon 541 was a conservative one which was also observed in the normal population and normal tissues of CML patients, it probably represents a polymorphic variation. Although samples of hair roots and leukemic cells from the chronic phase of one CML patient showed no abnormality, an abnormality at codon 541 (ATG --> CTG, Met --> Leu) was found only at blastic crisis (BC) of this case. In the case with the abnormality at codon 564, the mutation was detected only in a sample of leukemic cells collected at BC. To examine the biological consequence and biological significance of these abnormalities, murine KIT(L540) and KIT(K563) expression vectors were introduced into interleukin-3 (IL-3)-dependent murine Ba/F3 cells to study their state of tyrosine phosphorylation and their growth rate. Ba/F3 cells expressing KIT(WT), KIT(L540) and KIT(K563) showed dose-dependent tyrosine phosphorylation after treatment with increasing concentrations of recombinant mouse stem cell factor (rmSCF). The cells expressing KIT(L540) and KIT(K563) were found to have greater tyrosine phosphorylation than cells expressing KIT(WT) at 0.1 and 1.0 ng/ml of rmSCF. The Ba/F3 cells expressing KIT(K563) proliferated in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The Ba/F3 cells expressing KIT(L540)showed a relatively higher proliferative response to 0.1 ng/ml of rmSCF than the response of cells expressing KIT(WT). These mutations and in vitro functional analyses raise the possibility that the KIT abnormalities influence the white blood cell counts (P < 0.05) and survival (P < 0.04) of CML patients.

Establishment of a cell line with AML1-MTG8, TP53, and TP73 abnormalities from acute myelogenous leukemia.

Gene alterations accumulate during the progression of acute myelogenous leukemia (AML) to a malignant clone. Here, a new myeloid cell line, designated YSK-21, with the balanced t(8;21)(q22;q22) and the unbalanced der(1)t(1;17)(p36;q21), was established. YSK-21 grows well in a medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), granulocyte-macrophage colony-stimulating factor (rhGM-CSF), or interleukin-3 (rhIL-3). Molecular analysis using the reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) revealed that t(8;21)(q22;q22) resulted in an AML1-MTG8 fusion transcript. FISH and spectral karyotyping (SKY) in conjunction with G-banding analysis revealed a der(1)t(1;17)(p36;q21) chromosomal translocation, which appeared in the clone developed from the original leukemic cells. Molecular analysis of the TP73 gene on 1p36 and the TP53 gene revealed a deletion of one-allele in TP73 with partial demethylation of another allele in the initial clone of YSK, and a point mutation consisting of an A-->T substitution in codon 288 of the TP53 gene in the developed clone of YSK-21. YSK-21 cells, expressing aberrant AML1-MTG8, TP53, and TP73 protein molecules, may be useful for elucidating the pathophysiology of these aberrant proteins and for studying the der(1)t(1;17)(p36;q21) chromosomal translocation.

Initial symptoms of acute radiation syndrome in the JCO criticality accident in Tokai-mura.

A criticality accident occurred on September 30, 1999, at the uranium conversion plant in Tokai-mura (Tokai-village), Ibaraki Prefecture, Japan. When the criticality occurred, three workers saw a "blue-white glow," and a radiation monitor alarm was sounded. They were severely exposed to neutron and gamma-ray irradiation, and subsequently developed acute radiation syndrome (ARS). One worker reported vomiting within minutes and loss of consciousness for 10-20 seconds. This worker also had diarrhea an hour after the exposure. The other worker started to vomit almost an hour after the exposure. The three workers, including their supervisor, who had no symptoms at the time, were brought to the National Mito Hospital by ambulance. Because of the detection of gamma-rays from their body surface by preliminary surveys and decreased numbers of lymphocytes in peripheral blood, they were transferred to the National Institute of Radiological Sciences (NIRS), which has been designated as a hospital responsible for radiation emergencies. Dose estimations for the three workers were performed by prodromal symptoms, serial changes of lymphocyte numbers, chromosomal analysis, and 24Na activity. The results obtained from these methods were fairly consistent. Most of the data, such as the dose rate of radiation, its distribution, and the quality needed to evaluate the average dose, were not available when the decision for hematopoitic stem cell transplantation had to be made. Therefore, prodromal symptoms may be important in making decisions for therapeutic strategies, such as stem-cell transplantation in heavily exposed victims.

Chromosome aberrations in bone marrow cells from Japanese patients with thorotrastosis.

Exposure of bone marrow cells to alpha-particle radiation causes various types of chromosome abnormalities and hematological malignancies. We performed chromosome analysis of hematopoietic stem cells from the bone marrow of 52 Japanese patients with thorotrastosis and 21 age-matched controls. The frequency of cells with stable chromosome abnormalities was significantly higher in the patients with thorotrastosis. Further studies found 14 clonal chromosome aberrations in cells from 11 patients (21.2%); clones observed in the cells from 2 of these patients had high frequencies of chromosome abnormalities. In one case, 68 to 100% of the cells analyzed had a large partial loss in the short arm of chromosome 1 and a translocation between the short arms of chromosomes 2 and 3 [46,XY,1p-,t(2p+;3p-)]. The cells from the other patient contained a clone with partial loss of both the short and long arms of chromosome 5 (46,XX,5p-,5q-). The frequency of this clone has been constant for the last 15 years (6-24%). We also analyzed bone marrow mononuclear cells from 17 of the patients for mutations of the TP53 tumor suppressor gene (formerly known as p53). However, no mutation was found in any of the cells, including those from the 2 patients with abnormal clones. Moreover, repeated medical examinations showed no evidence of leukemia or myelodysplasia in these patients. Our study suggests that exposure of bone marrow cells to alpha-particle radiation may induce clonal chromosomal aberrations at a high frequency.

Establishment of a near-triploid human B-cell lymphoma cell line with t(14;18) and a p53 gene point mutation.

We report a rare large B-cell non-Hodgkin's lymphoma having a characteristic near-triploid cell population with add(17)(p22) and t(14;18)(q32;q21) translocation. We also established and characterized a new cell line (TK cell) derived from the present lymphoma. A codon 180 mutation (GAG --> GAT) in the p53 gene was detected. t(14;18)(q32;q21) was revealed juxtaposition of the bcl-2 and JH genes. Immunoprecipitation analyses of p53 and bcl-2 revealed that abnormality of the p53 protein and aberrant bcl-2 expression, which may protect cells from apoptosis, may be critical to the development of leukaemogenesis exhibiting near-triploid chromosomes.

Establishment of a cell line with variant BCR/ABL breakpoint expressing P180BCR/ABL from late-appearing Philadelphia-positive acute biphenotypic leukemia.

In acute leukemia (AL) with a late-appearing Philadelphia (la-Ph) translocation, it is unclear whether these translocations arise from the same molecular event as classical Ph translocations. In order to elucidate the molecular events of la-Ph and subsequent translocations of la-Ph leukemia, we performed molecular analysis on the complex rearrangements, in a cell line, MY, which was established from bone marrow mononuclear cells of a patient with a la-Ph acute biphenotypic leukemia. This la-Ph, expressing an acute lymphoblastic leukemia (ALL)-type BCR/ABL transcript, produces a novel P180BCR/ABL fusion protein reflecting deletion of 174 bases (58 amino acids) encoded by the a2 exon of the ABL gene. An immune complex kinase assay showed that this protein had autophosphorylation activity. Fluorescence in situ hybridization (FISH) in conjunction with G-banding analysis revealed that the initial der(9)t(9;22)(q34;q11) progressed to a der(9)(9pter-->9q34::22q11-->22q13::5q11.2 -->5q15:: 10q23-->10qter) by, first, a three-way translocation among the der(9)t(9;22)(q34;q11), chromosome 5, and the normal chromosome 22, and then a subsequent translocation with chromosome 10. Moreover, both the end-stage leukemic cells of the patient and the MY cell line had another translocation, t(X;12)(p11.2;p13). The 12p breakpoint was located near the ETV6 gene by analysis of pulsed-field gel electrophoresis, but transcription of ETV6 was unaffected. Tumorigenicity analysis indicated that an additional translocation, t(2;3)(p16;q29), may have caused a more malignant clone, because only MY cells with the t(2;3)(p16;q29) were capable of growing subcutaneously in nude mice within 40 days. The molecular events of leukemogenesis and leukemic progression in the present la-Ph AL occurred by accumulation of unique translocations. This cell line, MY, expressing a novel variant P180BCR/ABL protein with a deletion of the a2 exon of the ABL gene, may be useful for elucidating the pathophysiology of this fusion protein and for studying ETV6-related leukemogenesis and t(2;3), as well as the molecular mechanisms of the complex translocations.

Relation between microsatellite instability and N-ras mutation and duration of disease free survival in patients with acute leukemia.

BACKGROUND: Microsatellite instability (MIN) appears to be a novel molecular mechanism in carcinogenesis and is believed to reflect multiple replication errors. The authors analyzed MIN in de novo acute leukemia and its association with expression of the human MSH3 (hMSH3) gene and point mutations of the N-ras gene. The relation between MIN and disease free survival also was examined. METHODS: The authors studied 43 cases of de novo acute leukemia for MIN at 5 loci. They also examined expression of the hMSH3 gene using the reverse transcriptase-polymerase chain reaction method. DNA taken from 36 cases at diagnosis was examined for N-ras mutations. The Kaplan-Meier method was used to estimate patients' disease free survival. RESULTS: MIN at 1 locus was found in 7 of the 43 cases (16%) (MIN positive [MIN+] group). There was no correlation between MIN+ and decreased hMSH3 gene expression. Four of the 7 MIN+ cases (57%) had a mutation of the N-ras gene compared with only 1 of the 29 MIN- cases (3%). A correlation between N-ras mutations and MIN was detected (P < 0.003). The MIN+ patients had a shorter disease free survival than the MIN- patients (P < 0.03). CONCLUSIONS: MIN may be associated with N-ras mutation in some cases of de novo acute leukemia and influence the duration of disease free survival.

Minimal residual disease in acute myelogenous leukemia with PML/RAR alpha or AML1/ETO mRNA and phenotypic analysis of possible T and natural killer cells in bone marrow.

Here we studied minimal residual disease (MRD) of patients with acute myeloid leukemia (AML) who have PML/RAR alpha or AML1/ETO as well as the phenotypic analysis of lymphocyte subsets involved in antitumor immunity. Eight patients in long-term (LT; 3 to 15 years) and 15 patients in short-term (ST; up to 3 years) remission were studied. Using the reverse transcription-polymerase chain reaction (RT) assay, the limit of detection was 10(-5) to 10(-6) for PML/RAR alpha transcript and 10(-4) to 10(-5) for the AML1/ETO transcript. Simultaneously, T lymphocyte subsets and NK cells from the peripheral blood (PB) and bone marrow (BM) were investigated by flow cytometric analysis. Four of the eight patients in LT and 7 of the 15 patients in ST remission were MRD-positive. Although all MRD-positive patients in LT remission are still until now event-free, 3 of the 7 MRD-positive (MRD+) patients in ST remission soon relapsed. The total populations of CD4+, CD8+ and CD56+ [possible T-cell and natural killer (T/NK) populations] in the BM of ST patients and MRD+/LT patients were significantly (p < .01) low. The CD8+ CD28+ population showed the same tendency (p < .01-.02). The T/NK subsets in the BM of MRD-negative (MRD-) LT (MRD-/LT) patients showed similar numbers of cells as normal volunteers. Basically, the total percentage of the CD4+, CD8+ and CD56+ cell populations in the BM was increased and in the following order: MRD-/LT patients, normal volunteers, MRD+/LT patients and MRD+ or -/ST patients. The percentages of the T/NK-cell subsets in the PB were not significantly different among these groups. Thus, the difference of the possible T/NK-cell phenotype in the BM may strongly influence clinical and molecular remission. These results still remain to be confirmed by further studies of the functional anti-tumor immunity of T/NK cells of AML in remission.

Highly scattering intralipid-10% assisted lasing from microdroplets with acridine orange dye.

One order or greater of magnitude enhancement of lasing emission is confirmed experimentally from liquid microdroplets of Acridine Orange dye mixed with the fat emulsion Intralipid-10% suspension as highly scattering media, compared with pure dye-doped droplets. This novel method that makes use of a high-gain laser dye soft scatter microsystem allows for a wide range of lasing of microdropslets. Originally without lasing pure dye droplets enabled one to lase with a well-defined threshold and an appreciably increased emission intensity in suitable conditions. Spectral characteristics and emission peak intensities from these microdroplets are measured quantitatively as a function of volume content of Intralipid-10% solutions.

Heterogenous expression of bcr-abl fusion mRNA in a patient with Philadelphia-chromosome-positive acute lymphoblastic leukaemia.

We performed molecular and cytogenetic analysis on a 56-year-old woman with Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) having two types of major and minor bcr-abl (M-bcr-abl, m-bcr-abl/fusion mRNA at diagnosis. In the course of her disease, unexpected heterogenous bcr-abl fusion mRNA was detected by sequential analysis using the reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR analysis showed both M-bcr-abl (b2-a2 type) and m-bcr-abl at diagnosis. Although b2-a2 type M-bcr-abl disappeared after complete remission (CR) was achieved with intensive induction chemotherapy, expression of both m-bcr-abl and a new type of M-bcr-abl mRNA (b1-a2 type), which may have been produced through splicing out of exon b2, was detected in the early stage of CR. The leukaemic cells contained these heterogenous bcr-abl mRNAs in both the CR and relapsed state, and showed more stable expression of the m-bcr-abl type mRNA than that of the b2-a2 type. These findings of heterogenous bcr-abl mRNA due to alternative or missplicing during the disease course in the present ALL case may provide important evidence of disease progression.

Possible transforming activity of interferon regulatory factor 2 in tumorigenicity assay of NIH3T3 cells transfected with DNA from chronic myelogenous leukemia patients.

Little is known about the transforming gene identified in the genomic DNA of chronic myelogenous leukemia (CML) by the tumorigenicity assay. To detect a new transforming gene of CML, we re-investigated the transforming activity of interferon regulatory factor (IRF)-1 and -2 genes in the tumorigenicity assay of NIH3T3 cells transfected with genomic DNA of leukemic cells from 15 patients with CML (12 patients in the chronic phase, one in the blastic phase and two in both phases). We detected the functionally active IRF-2 gene only in the tumor DNA from two CML patients in the blastic phase. We did not detect integration of the IRF-1 gene in the DNA of any tumors derived from the CML patient samples, and also we detected no expression of human IRF-1 mRNA. Thus, NIH3T3 cells may have been transformed due to integration of the functionally active IRF-2 gene from CML patients in the blastic phase. We surmise that there is a possibility that the IRF-2 gene may be involved in the evolution of the blastic phase of CML.

Lasing behavior in a liquid spherical dye laser containing highly scattering nanoparticles.

With the addition of scattering nanoparticles to dye-doped spherical droplets, lasing has been observed with well-defined thresholds in input-output data. One-order or more magnitude enhancement of peak intensity from droplet emission has been obtained with certain (optimum) additive scattering particles compared with nonadditive scattering particles (neat dye-doped droplets). Characteristics of input-output intensities, emission spectra (with wavelength shifts), and spectral linewidths are reported experimentally, depending on additive quantities of scattering nanoparticles.

Dual rearrangement of immunoglobulin and T-cell receptor genes in a case of splenic lymphoma with villous lymphocytes.

We report here a case of "splenic lymphoma with villous lymphocytes" (SLVL) which exhibited both B- and T-cell phenotypes and genotypes. The patient was a 73-year-old man. Physical examination revealed splenomegaly and lymphadenopathy. The white blood cell count was 55.2 x 10(9)/L with 70.5% atypical lymphocytes, having cytoplasmic villi, characteristic of SLVL. The atypical cells infiltrated both the red and white pulps. Immunological analysis of the peripheral leukocytes showed both B- and T-cell phenotypes (CD5,CD11c,CD19,CD20,HLA-DR, SmIgM and lambda positive). DNA analysis revealed a dual rearrangement of the immunoglobulin heavy chain gene and T-cell receptor beta gene. SLVL has been identified as a B-cell leukemia with a relatively benign clinical course. This case had both B- and T-cell pheno- and genotypes with a progressive course. To the best of our knowledge, no case of SLVL with dual genotypes has ever been reported.