Summary.A novel GM-CSF-dependent myeloid cell line, OHN-GM, was established from a patient who developed acute myelogenous leukaemia (AML) as a consequence of myelodysplastic syndrome (MDS). As the patient had previously received cytotoxic chemotherapy for Hodgkin's disease, the MDS and AML were probably related to such therapy. Sequential karyotypic analysis established a del(5q) as the initial cytogenetic abnormality. Additional alterations, including t(10;13)(q24;q14), had developed subsequently during disease progression. Southern blot analysis of OHN-GM cells suggested deletion of one allele of the IRF-1 gene, although no aberrant transcripts were detected. Fluorescence in situ hybridization analysis revealed the deletion of the Rb gene due to the t(10;13)(q24;q14) translocation, and Western blot analysis demonstrated the absence of Rb protein in OHN-GM cells. Finally, the OHN-GM cells exhibited two missense point mutations in highly conserved regions of the p53 gene. These observations suggest that a multistep process, involving alterations of Rb and p53 genes, may have contributed to the patient's disease development and progression. To our knowledge, OHN-GM is the first cell line derived from a therapy-related AML. These cells may aid the investigation of leukaemogenesis as well as the biology of secondary leukaemia.

Summary.A novel human EBV-negative B-cell line, designated DOBIL-6, was established from a patient with nonsecretary myeloma. The DOBIL-6 cell has cytoplasmic g protein and expresses CD19, 20, 38, 45RO, VLA-4 and PCA-1 antigens, but lacks CD10, 45RA and VLA5 antigens. Chromosome analysis showed that DOBIL-6 cells had many complex structural abnormalities, including t(11;14) (q13;q32), which were consistent with that of the fresh tumour cells. Interestingly, abundant interleukin-6 (IL-6) and parathyroid hormone-related protein (PTHrP) accumulated in the culture supernatant of DOBIL-6 cells. Hypercalcaemia and splenomegaly associated with plasma cell proliferations which resulted in the expansion of the light zones in the follicles were observed in DOBIL-6 transplanted nude mice. RT-PCR analysis detected mRNA for PTHrP, and IL-6 as well as its receptor (GP80) in DOBIL-6 cells.Treatment of the DOBIL-6 cells with neutralizing anti-IL-6 antibody inhibited their growth in a dose-dependent manner, whereas the addition of exogenous IL-6 stimulated it in serum-depleted conditions. These findings suggest that both IL-6 and PTHrP are produced in DOBIL-6 cells, and that IL-6 promotes its growth by an autocrine mechanism. Since IL-6 is known to stimulate not only the growth of B-cell neoplasms but also osteoclastic bone resorption by cooperating with PTHrP, this simultaneous production of IL-6 and PTHrP might be synergistically linked and play a role in the development of hypercalcaemia of the patient. The DOBIL-6 cell is a useful tool to clarify the mechanism of hypercalcaemia associated with mature B-cell neoplasms.

A newly established human lymphoma cell line (OZ) has the t(14;18)(q32;q21) translocation and expresses large amounts of Bcl-2 compared to CCRF-CEM cells. VP-16 (40 micrograms/mL), a promising agent against lymphoma, caused DNA fragmentation (26.9% of total DNA) typical for apoptosis at 6 h in CCRF-CEM cells, but no significant changes in OZ cells until 24 h after the addition of VP-16. However, coincubation with calphostin C (0.2 microgram/mL), a protein kinase C (PKC) inhibitor, induced DNA fragmentation in VP-16-treated OZ cells (13.5% of total DNA) at 6 h after the treatment. Simultaneous immunoblot analysis revealed that this induction of apoptosis coincided with the downregulation of serine-phosphorylated Bcl-2 (13% of control cells). By contrast, apoptosis induced by VP-16 in CCRF-CEM cells was attenuated by the addition of 0.5 microM phorbol 12-myristate 13-acetate, a potent PKC stimulator. These observations suggest that Bcl-2 function is partly regulated by phosphorylation/ dephosphorylation mechanisms of the PKC system, and that phosphorylated Bcl-2 in lymphoma cells may play a role in the prevention of apoptosis.

Summary. A novel cell line, designated OHK, was established from ascites of a 59-year-old Japanese woman with diffuse large B-cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed±Sternberg-like giant cells. On ultrastructural examination, rich intermediate ®laments, and well-developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell-associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposi's sarcomaassociated herpesvirus and Epstein±Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage-colony-stimulating factor (M-CSF), granulocyte-CSF, interleukin 6 and transforming growth factor b1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.

The novel human pre-B cell line OZ was established from a patient with an aggressive form of non-Hodgkin's lymphoma. Karyotypic analysis of both the primary tumour and OZ cells revealed several marker chromosomes, including the t(14;18)(q32;q21) translocation, which involves the Bcl-2 gene, and alterations on chromosome 17p. Southern blot analysis found identical rearrangements in the 5' region of Bcl-2 gene in the primary tumour and OZ cells. Homozygous deletions of the p15INK4B and p16INK4A genes, however, were present only in OZ cells. Western blot analysis detected aberrant small molecular-weight p53 proteins in both cell types. In addition, OZ cells no longer expressed the CD20 antigen. These findings suggest that Bcl-2 gene rearrangement and aberrant p53 expression resulted in the original B-cell tumour. A subsequent transforming event involving the p15INK4B and p16INK4A genes may have generated more immature cells with a growth advantage during in vitro culture. The genetic alterations involving p53, p15INK4B, and p16INK4A may be implicated in the aggressive form of t(14;18)(q32;q21)-bearing tumours and their poor prognosis.