Acquired cytotoxic activity of CD8+ HIV-1IIIB carrier immature T cell clones specific to CD4+CD8+ (parental) human T cell clones and normal thymocytes.

By infecting human leukemia cell lines in vitro with HIV-1IIIB' a number of HIV-1 carrier clones were generated. Among them, 5 of 13 CD8+ HIV-1 carrier T cell clones were shown to acquire a rapid cytotoxic activity (within 1 hour) specific to TdT+CD4+CD8+ immature T cells including normal thymocytes. This novel cytotoxic reaction, without requiring virus infection and indicating a rapid T cell precursor elimination during active lymphopoiesis, suggests a mechanism responsible for mature CD4+ T cell depletion in HIV-1 infected individuals.

In vitro study using leukemia cell line panel to demonstrate rapid thymic T cell death due to contact with HIV-1 carrier cell clones.

A rapid (within 1 h) and profound cytotoxic cell death of immature TdT+CD4+CD8+ and/or TdT+CD4+ thymic T cell type leukemia cell lines, and of normal thymocyte populations rich in TdT+CD4+CD8+ cells was induced by contact with some human immunodeficiency virus type-1 (HIV-1) carrier T cell clones. This cytotoxic reaction, without requiring a complete viral replication cycle in the thymic T cells, did not occur in any mature CD4+CD8+ and/or CD4+ T cells which are otherwise permissive for virus infection. Although it was not an antigen-specific cytotoxic reaction, the rapid and profound thymic T cell destruction was shown, at the individual clonal level, to be triggered specifically by the binding of CD4 molecules on thymic T cells with gp120/gp160 on HIV-1 carrier clones. The present study suggesting direct elimination of immature T cells by contact with some HIV-1-infected T cells, may provide a novel insight into the mechanism responsible for the mature CD4+ T cell depletion in HIV-1 infection.

A novel ALL-L3 cell line, BALM-16, lacking expression of immunoglobulin chains derived from a patient with hypercalcemia.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-16, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) in relapse. As with the original leukemia cells, the established line was negative for both cell surface and cytoplasmic immunoglobulin (Ig) chains. Absence of Ig expression was confirmed by Western blotting. Southern blot analysis demonstrated homozygous deletion of the C kappa gene, germ line configuration of the C lambda and rearrangement of IgJH genes. Cytogenetic analysis of both leukemic bone marrow and BALM-16 cells showed the t(8;22)(q24;q11) abnormality which is specifically associated with ALL-L3 and Burkitt lymphoma. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which showed parathyroid hormone-related peptide (PTHrP) mRNA detected by reverse-transcriptase polymerase chain reaction. However, PTHrP production was not detected in the culture supernatant. The established cell line, BALM-16, could provide a useful material for analyzing the lack of Ig expression and of clarifying the pathogenesis of this type of B cell malignancy.

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23).

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.

Establishment and characterization of a novel ALL-L3 cell line (BALM-18): induction of apoptosis by anti-IgM and inhibition of apoptosis by bone marrow stroma cells.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-18, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) at diagnosis using bone marrow stroma cells (BST) as feeder cells. The primary leukemia cells did not grow without feeder cells. As with the primary leukemia cells, BALM-18 showed an immunophenotype of Burkitt's lymphoma group I [CD10+, CD20+, CD23-, CD39-, CD77+] and carried the t(8;14)(q24;q32) chromosomal abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. It also revealed a significantly low level of bcl-2 protein. Strikingly, anti-human IgM antibody did induce apoptosis in induction experiments. However, it was reversed by the addition of anti-CD40 antibody or BST cells, whereas the culture supernatant of the stroma cells did not show any effect on the inhibition of apoptosis. BALM-18 may be useful for analyzing both the mechanisms of anti-IgM induced apoptosis and signaling during the inhibition of apoptosis by CD40 or BST cells.

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

Human bone marrow stroma-dependent myeloma sister cell lines MOLP-6 and MOLP-7 derived from a patient with multiple myeloma.

Human bone marrow stroma (BST)-dependent myeloma sister cell lines MOLP-6 and MOLP-7 were established from the peripheral blood of a multiple myeloma (MM) patient with IgA kappa type MM (stage IIIB). The growth of the cell lines is constitutively dependent on BST cells; none of the cytokines tested nor the culture supernatant of the BST cells could support the growth. Both cell lines showed typical plasma cell morphology with abundant cytoplasm and one to four nuclei under Wright staining. The immunoprofiles of MOLP-6 and MOLP-7 correspond to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) chains, a heavy and kappa light chains, CD9, CD28, CD40, CD44, CD45, CD56, and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte associated markers. Both cell lines also expressed adhesion molecules including HCAM (CD44), VLA-4 (CD49d/CD29), VLA-6 (CD49f/CD29), ICAM-1 (CD54), NCAM (CD56), LFA-3 (CD58) and L-selectin (CD62L). The doubling time of MOLP-6 and MOLP-7 was 48 and 168 hours, respectively. In addition to this growth characteristic, the maximum cell density of each cell line was obtained at 1.7 x 10(6) cells/ml and 9.7 x 10(5) cells/ml, respectively. The characteristics of each cell line may reflect intraclonal variation of the proliferative capacity. The MOLP-6 together with the MOLP-7 sister will be useful model systems for the investigation of the biology of myeloma.

Myeloperoxidase positive acute lymphoblastic leukemia cell lines, NALM-30, NALM-31 and NALM-32, carrying Philadelphia chromosome with biphenotypic characteristics.

We established three sister cell lines, NALM-30, NALM-31 and NALM-32, with biphenotypic features carrying myeloperoxidase mRNA and protein with complex Philadelphia (Ph) chromosome, t(9;22;10)(q34;q11;q22), from a patient with Ph-positive acute leukemia in relapse. Epstein-Barr virus nuclear antigen was negative. The morphological appearance of the cell lines is that of immature lymphoid cells. Expression of myeloid- and lymphoid-associated surface membrane antigens on these cells was detected allowing for the classification of "biphenotypic" leukemia. Immunophenotypically, the established cell lines reported here fulfill the European Group for the Immunological Characterization of Leukemias (EGIL) criteria for B-lineage derivation, however, surface and cytoplasmic immunoglobulin chains were negative. Whereas TGF-beta R (CD105), MCSFR (CD115), SCFR (CD117), IL-4R/IL-13R (CD124) and IL-6R (CD126) were not expressed, the cell lines were mostly positive for IFN-gamma R (CD119), IL-7R (CD127) and FLT-3R (CD135). The NALM-30, NALM-31 and NALM-32 cell lines together with their serial sister cell lines NALM-27 and NALM-28 which were established from the same patient at diagnosis provide unprecedented opportunities for studying a multitude of biological aspects related to normal and neoplastic immature B-lymphocytes.

Inhibitory effect of chlorophyll on the genotoxicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2).

Genotoxicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) on Drosophila was suppressed by chlorophyll. Using the wing hair spot test, we found that the formation of mutant hairs in adult flies as a result of feeding them with Trp-P-2 in their larval stage was efficiently inhibited by coadministration of chlorophyll. The decrease in the spot frequencies was dependent on the dose of chlorophyll, and at the highest dose used, where the ratio in weight of Trp-P-2 to chlorophyll was 1:80, a complete prevention of the small single-spot formation was observed. A similar inhibitory effect was detected for chlorophyllin, the chromophore of chlorophyll. In the studies to investigate the mechanism of inhibition, we observed that the mutagenicity of 3-hydroxy-amino-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NHOH)], the metabolically activated form of Trp-P-2, in Salmonella typhimurium TA98 was suppressed effectively with chlorophyll and chlorophyllin. We also found that chlorophyll and chlorophyllin can produce complexes with Trp-P-2 and Trp-P-2(NHOH). A straightforward mechanism of these inhibitions is that Trp-P-2 [and Trp-P-2(NHOH)] becomes no longer available to organisms on forming the chlorophyll complex.

Expression of the ryanodine receptor isoforms in immune cells.

Ryanodine receptor (RYR) is a Ca(2+) channel that mediates Ca(2+) release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type 1 RYR (RYR1) was expressed in CD19(+) B lymphocytes, but less frequently in CD3(+) T lymphocytes and in CD14(+) monocytes. Type 2 RYR (RYR2) was mainly detected in CD3(+) T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha (MIP1alpha) or TGF-beta. Type 3 RYR (RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was not associated with specific cell lineage. We showed that the RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca(2+) release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant induction of RYR mRNA with Ca(2+) channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no effect on Ca(2+) release, whereas 4CmC (<400 microM) caused Ca(2+) release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha, or TGF-beta. Our results demonstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and TGF-beta suggests roles in regulating Ca(2+)-mediated cellular responses during the immune response.

Human bone marrow stroma-dependent cell line MOLP-5 derived from a patient in leukaemic phase of multiple myeloma.

The novel multiple myeloma (MM) cell line MOLP-5 and its homologous sister cell line B407, a lymphoblastoid cell line (LCL), were established from the peripheral blood of a 71-year-old Japanese patient with Bence-Jones kappa-type multiple myeloma (stage IIIB with hyperammonaemia and hypercalcaemia). The growth of MOLP-5 cells is constitutively dependent on bone marrow stroma (BST) cells; none of the cytokines tested nor the culture supernatant of the bone marrow stroma cells could support the growth of MOLP-5. Wright-Giemsa-stained MOLP-5 cells showed typical plasma cell morphology with abundant cytoplasm and one to three nuclei. The immunoprofile of MOLP-5 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) kappa light chain, CD28, CD29, CD38, CD40, CD44, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Ig and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. Interleukin 6 (IL-6) receptor mRNA was found in the reverse transcriptase polymerase chain reaction (RT-PCR) analysis. IL-6 and IL-10 could induce cellular proliferation in short-term induction experiments. IL-6 or IL-10 production was not detected by specific enzyme-linked immunoabsorbent assay (ELISA). MOLP-5 cells expressed parathyroid hormone-related protein (PTHrP) at the mRNA level. Cytogenetic analysis showed the typical t(11; 14) chromosome abnormality. The novel MOLP-5 cell line together with the B407 B-LCL sister line will be useful model systems in the investigation of the biology of MM.