Classification and biological nature of established human hematopoietic cell lines.

Over 200 established human hematopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. (1) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus (EBV), polyclonal derivation, differentiation for immunoglobulin production (secretion) and their diploids. (2) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with EBV, variability in the expression of Ig synthesis (no production, or membrane bound Ig, or secretion) and aneuploidy. (3) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of EBV and aneuploidy. (4) The leukemia cell line (MOLT 4) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typical surface ultrastructure, absence of EBV and absence of immunoglobulin production, Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. We conclude that EBV-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells;     

Surface glycoprotein patterns of normal and malignant human lymphoid cells. II. B cells, B blasts and Epstein-Barr virus (EBV)-positive and -negative B lymphoid cell lines.

Surface glycoproteins of normal human B lymphocytes, B blasts and various types of lymphoid cell lines were labelled by the galactose oxidase-tritiated sodium borohydride method. The labelled glycoproteins were separated by polyacryUmide slab gel electrophoresis in the presence of sodium dodecy! sulfate and visualized by modified autoradiography (fluorography). The battery of examined hematopoietic cell lines included Epstein-Barr virus (EBV) carrying lines of proven malignant origin (Burkitt's lymphoma) and presumed non-neoplastic origin (lymphoblastoid cell lines), and EBV-genome-negative lymphocytic lymphoma, histiocytic lymphoma, myeloma and myeloid leukemia lines. The presence of possible EBV-associated surface glycoproteins, detectable by the labelling method, was studied by use of two EBV-negative cell lines (BJAB and Ramos) and their EBV-converted sublines. The six Burkitt lymphoma and three lymphocytic lymphoma lines had all the basic surface glycoprotein pattern of resting B cells and, in addition to individually distinct bands, two characteristic pairs of glycoproteins sf apparent molecular weights of 87,000/85,000 and 71,000/69,OOO. These glycoproteins were not detected on the normal B celts, B blasts or non-neoplastic lymphoblastoid lines. Neither were they found on the other types of neoptastic line. No consistent difference in the surface glycoprotein patterns was detected between the EBV-genome-negative and EBV-con-verted BJAB and Ramos sublines. The glycoprotein pattern of the six lymphoblastoid lines resembled that of the B blasts. The histiocytic lymphoma, myeloma and leukemia lines all had distinct patterns. These results confirm that the Burkitt's lymphoma and the lymphoblastoid cell lines represent two biologically distinct EBV-carrying B lymphoid celts and that the galactose oxidase NaB[³H]₄ surface labelling technique can be used as a reliable molecular mapping method to distinguish between these two and other types of lymphoid cell lines.     

Cell surface glycoprotein changes in Epstein-Barr virus-positive and -negative human hematopoietic cell lines.

It has previously been shown that differential fucose labelling of many normal and homologous tumor cells, followed by proteolytic release and degradation, yields glycopeptides which upon gel filtration shown an increase in fast-eluting glycopeptides for the tumor cells. This technique has now been applied to cell-surface glycoproteins of different human hematopoietic cell lines. These lines included Epstein-Barr virus (EBV)-carrying lymphoblastoid cell lines of presumed non-neoplastic origin, and malignant EBV-genome-positive Burkitt lymphoma and EBV-negative non-Burkitt lymphoma, leukemia and myeloma lines. As compared with normal peripheral lymphocytes, both the lymphoblastoid type of cell lines and the different types of lines of proven malignant ancestry contained the fast-eluting glycopeptides on their cell surface with very few exceptions. It is therefore concluded that (I) malignant conversion of human lymphoid cell in vivo is commonly, but not obligatorily, associated with a specific change in the composition of the fucosyl glycopeptides, and (2) EBV infection of B lymphocytes does not lead only to the well-documented immortalization in vitro but also, as a rule, to the same type of alteration in fucosyl glycopeptides as was demonstrated for the neoplastic cell lines. It proved possible to distinguish several categories of hematopoietic cell lines due to the effect that pretreatment of the glycopeptides with neuraminidase or mild acid exerted on their subsequent chromatographic behavior.     

Tumorigenicity of human hematopoietic cell lines in athymic nude mice.

Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines.     

Establishment and characterization of two human myeloma cell lines secreting kappa light chains

A small number of human myelomas have been established as long term cultured cell lines. We report the characteristics of two new cell lines, designated SK-MM-1 and SK-MM-2, derived from 73 attempts to culture myeloma specimens. Both cell lines were grown from myeloma patients with hypogammaglobulinemia, kappa light chain proteinuria, and plasma cell leukemia. SK-MM-1 and SK-MM-2 had a plasmacytoid morphology, grew in RPMI complete medium with doubling times of 32 and 60 hr, respectively, and did not express Epstein-Barr virus nuclear antigen. Both cell lines secreted kappa light chains (0.9 and 1.1 micrograms/10(6) cells/ml per 48 hr for SK-MM-1 and SK-MM-2, respectively) but no heavy chains. SK-MM-1 and SK-MM-2 expressed the pan-B cell marker B1 and the late B cell/plasma cell marker BL3. In addition, SK-MM-2 expressed late B cell/plasma cell markers OKT10 and PCA-1. Neither cell line expressed T lymphocyte, myeloid, or early B lymphocyte markers. The presence of distinctive kappa and heavy chain gene rearrangements supported the clonal origin of both cell lines from kappa light chain-producing B cells. The two cell lines were markedly aneuploid and both carried a 14q+ marker chromosome. Human myeloma cell lines lacking heavy chain secretion may be useful to elucidate mechanisms of immunoglobulin gene regulation and to construct human-human hybridomas.     

Promising preclinical activity of 2-methoxyestradiol in multiple myeloma.

PURPOSE: 2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol metabolism, has demonstrated activity against tumor cell lines and can inhibit angiogenesis. There are limited treatment options for patients with multiple myeloma (MM) who relapse after high-dose therapy and stem cell transplantation. We studied the preclinical activity of 2ME2 as a therapeutic agent for myeloma. EXPERIMENTAL DESIGN: Five established myeloma cell lines as well as primary plasma cells from patients with MM were exposed to 2ME2 at various concentrations. We evaluated the activity of the drug to inhibit cell replication and induction of apoptosis in vitro as well as the ability of the drug to inhibit myeloma tumor xenograft growth in severe combined immunodeficient mice. RESULTS: 2ME2 inhibited tritiated thymidine uptake in all myeloma cell lines tested in a dose-dependent fashion and induced G(2)-M phase cell cycle arrest. The drug induced apoptosis in all cell lines tested and in half of the primary plasma cells evaluated in a dose-response manner. Forty-eight h after drug exposure, a large proportion of the cells were dead by propidium iodide staining. Injection of the drug i.p. suppressed myeloma tumor xenograft growth, and the effect was sustained after cessation of therapy. CONCLUSIONS: 2ME2 has significant activity against myeloma cell lines and primary myeloma cells both in vitro and in an animal model. Clinical trials are required to evaluate its activity in patients with MM.     

Arsenic trioxide and melarsoprol induce apoptosis in plasma cell lines and in plasma cells from myeloma patients

Recent data have renewed the interest for arsenic-containing compounds as anticancer agents. In particular, arsenic trioxide (As2O3) has been demonstrated to be an effective drug in the treatment of acute promyelocytic leukemia by inducing programmed cell death in leukemic cells both in vitro and in vivo. This prompted us to study the in vitro effects of As2O3 and of another arsenical derivative, the organic compound melarsoprol, on human myeloma cells and on the plasma cell differentiation of normal B cells. At pharmacological concentrations (10(-8) to 10(-6) mol/L), As2O3 and melarsoprol caused a dose- and time-dependent inhibition of survival and growth in myeloma cell lines that was, in some, similar to that of acute promyelocytic leukemia cells. Both arsenical compounds induced plasma cell apoptosis, as assessed by 4',6-diamidino-2-phenylindole staining, detection of phosphatidylserine at the cell surface using annexin V, and by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. As2O3 and melarsoprol also inhibited viability and growth and induced apoptosis in plasma-cell enriched preparations from the bone marrow or blood of myeloma patients. In nonseparated bone marrow samples, both arsenical compounds triggered death in myeloma cells while sparing most myeloid cells, as demonstrated by double staining with annexin V and CD38 or CD15 antibodies. In primary myeloma cells as in cell lines, interleukin 6 did not prevent arsenic-induced cell death or growth inhibition, and no synergistic effect was observed with IFN-alpha. In contrast to As2O3, melarsoprol only slightly reduced the plasma cell differentiation of normal B cells induced by pokeweed mitogen. Both pokeweed mitogen-induced normal plasma cells and malignant plasma cells showed a normal nuclear distribution of PML protein, which was disrupted by As2O3 but not by melarsoprol, suggesting that the two arsenical derivatives acted by different mechanisms. These results point to the use of arsenical derivatives as investigational drugs in the treatment of multiple myeloma.     

Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro

Malignant plasma cells exert osteoclast-like activity in vitro. We investigated the function of the calcitonin (CT) receptor (R) on myeloma cells from patients and in myeloma cell lines. Primary myeloma cells expressed high CTR levels whereas the cell lines uniformly exposed the CTR-2 variant expressed by osteoclasts. Treatment of myeloma cell lines with CT modified the intracellular Ca(2+) and cAMP levels, suggesting the activation of both PKC and PKA pathways, and abrogated their bone resorptive property as erosive pits on osteologic substrates. Thus, the expression, sensitivity and function of CTR-2 in myeloma cells emphasize their osteoclast-like behavior in vitro.     

Granulocyte colony stimulating factor-producing multiple myeloma associated with neutrophilia

We report a case of IgG-kappa multiple myeloma associated with neutrophilia (WBC 31,300/microl, neutrophil 90.5%). Interestingly, the serum level of granulocyte colony stimulating factor (G-CSF) in this patient was elevated to 1,500 pg/ml (normal range: 5.78-27.5). Plasma cells were 35% in the bone marrow and were strongly stained with anti-G-CSF antibody. To directly study the production of G-CSF from plasma cells in this patient, CD138 positive plasma cells were purified from bone marrow of multiple myeloma patients by magnetic sorting. The expression of G-CSF mRNA was observed in CD138 positive plasma cells from this myeloma patient with neutrophilia by RT-PCR. In contrast, the expression of G-CSF mRNA was not detected in CD138 positive plasma cells from the other multiple myeloma patients without neutrophilia and 4 human myeloma cell lines (HS-Sultan, IM9, RPMI8226, U266) by RT-PCR. After the CD138 positive plasma cells were cultured in vitro for 48 h, the production of G-CSF protein was confirmed (71.8 pg/ml) in the supernatant by ELISA. These results indicated plasma cells of this myeloma patient directly produced G-CSF and that this was the primary cause of neutrophilia.     

Recent progress in multiple myeloma

Multiple myeloma is recognized as a neoplasm of phenotypically mature plasma cells which produces a variety of clinical symptoms related both to tumour infiltration of the bone marrow and cytokine production. The latter results in bone disease and a complex interactive network between plasma cells, marrow stromal cells and other hematopoietic cells. This serves to sustain the myeloma proliferative pool and promote maturation and secretion of monoclonal immunoglobulin. Whereas the recognizable tumour cells in myeloma are the most mature B cells, early lymphoid cells are involved in the disease and probably represent the proliferative pre-plasma cell compartment. The definition of the myeloma‘stem cell’ remains controversial, but our understanding of early pre-plasma cell differentiation in multiple myeloma has been aided by studies on normal non-neoplastic equivalents. Techniques like high resolution flow cytometry, flow cytometric DNA analysis and improvements in our ability to obtain karyotypic data in multiple myeloma will improve our understanding of myeloma cell biology, hopefully yielding new prognostic information. Finally, improvements in assessing prognosis will help identify patients whose survival with standard therapy is limited and how may require innovative or aggressive treatment protocols. These individuals must be separated from patients who either require no initial therapy or who are likely to have good outcomes with standard approaches.     

Establishment and characterization of a human histiocytic lymphoma cell line (U-937).

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.     

Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance

Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.     

CD19 expression and growth inhibition of tumours in human multiple myeloma

Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow (BM). CD19 is a hallmark differentiation antigen of the B cell lineage and positively regulates antigen receptor signal transduction in mature B cells. We have previously shown that malignant plasma cells (myeloma cells) isolated from the MM patients lack the CD19 expression, while non-malignant plasma cells isolated from the healthy donors do express the CD19 antigens. It is also intriguing that there exists both CD19- and CD19+ plasma cells in some cases in pre-myeloma states including monoclonal gammopathy of undetermined significance (MGUS). It indicates that MGUS is usually composed of phenotypically non-malignant (CD19+) and malignant (CD19-) plasma cells. Furthermore, we recently demonstrate that, expression of the CD19 gene markedly inhibits the proliferation of human myeloma cell lines in vitro, and exhibits the reduced tumorigenicity in vivo and no anchorage-independent growth in vitro of a tumorigenic myeloma cell line. This inhibitory effect might result from the CD19-mediated intracellular signals because it is not observed in cells expressing the mutant CD19, which lacks the cytoplasmic domain. In this review, we suggest that loss of CD19 in MM could contribute to the proliferative advantage of the malignant plasma cell clones in this disease. Furthermore, we propose the usefulness of the phenotypic analysis of plasma cells in human plasma cell dyscrasia as a new diagnostic tool, and the CD19 gene as a potential target for the gene therapy in MM.     

Establishment of a new human myeloma cell line (U-2030) and selection of a hat-sensitive subline

We have established a new human myeloma cell line from the pleural effusion of a patient with an IgA lambda myeloma, using special tissue culture conditions and selection procedures to prevent the outgrowth of contaminating Epstein-Barr virus (EBV)-carrying normal B-lymphoblastoid cells present in the explant. The myeloma cell line, U-2030, is aneuploid and EBNA-negative and has morphological features, reactivity with cytochemical markers and cell-surface antigen expression typical of plasmablasts. The cell line thus appears to be representative of the malignant clone in vivo. However, functionally the line is a non-Ig-producer and must therefore be derived from a non-secretory variant cell present within the highly aneuploid myeloma cell clone in vivo. The U-2030 differs from previously established human myeloma cell lines in that it has a comparatively high growth rate, is clonable and can be made HAT-sensitive relatively easily. This, together with the facts that it is a non-Ig-producer and mycoplasma-free, suggests that the 6-thioguanine-resistant, HAT-sensitive subline, U-2030 TG, derived from this cell line may be used as a malignant fusion partner for the production of human-human hybridomas. An EBV-carrying lymphoblastoid cell line (U-2031) was also established. This line was diploid and had all the phenotypic properties of lymphoblastoid lines established from normal individuals.     

Studies of four new human myeloma cell lines

We describe the establishment and studies of four myeloma cell lines that were derived from 2 young individuals with plasma cell leukaemia (Karpas 25 and 1272) and from 2 older persons with multiple myeloma (Karpas 417 and 929). The cultured cells have the ultrastructural appearance of plasma cells with abundant rough endoplasmic reticulum (RER). Their phenotypic profile conform with that of malignant plasma cells. Karyotype analysis revealed that each cell line is unique and all are hyperdipoide with complex aberrant chromosomes. FISH analysis revealed cryptic translocations affecting the IGH locus in 14q32 of 2 of these cell lines. Using R- and G-banding numerous other chromosomal abnormalities were recorded and illustrated in each of the 4 cell lines.     

Cytokine network in human multiple myeloma.

In multiple myeloma (MM), an overproduction of IL-6, indicated by increased plasma C-reactive protein levels, is found in 37% of MM patients at diagnosis and is associated with disease aggressiveness, myeloma-cell proliferation, and poor prognosis. IL-6 is produced by the tumoral environment mainly and not by myeloma cells themselves. IL-6 is a major growth factor for malignant plasmablastic cells in vitro, and it is possible to reproducibly obtain IL-6-dependent myeloma-cell lines. Moreover, anti-IL-6 therapies in patients with terminal disease block myeloma-cell proliferation in vivo. The myeloma-cell growth factor activity of IL-6 is probably the consequence of IL-6 being a growth factor for normal plasmablastic cells. Hematopoietic cytokines (GM-CSF, IL-3, IL-5, G-CSF) synergize with IL-6 to support myeloma-cell proliferation. IFN-alpha and TNF induce an autocrine production of IL-6 in myeloma-cell lines and make possible the autonomous growth of these cell lines. On the contrary, IFN-gamma completely inhibits the IL-6-mediated myeloma-cell proliferation. The identification of some major cytokines involved in the control of the myeloma clone has immediate therapeutic implications, because some of these cytokines are, or might be, used in the treatment of patients with MM.     

Cytoplasmic receptor levels and glucocorticoid response in human lymphoblastoid cell lines.

The cytolethal response to treatment with prednisolone was investigated in vitro in eight human lymphoblastoid cell lines containing varying concentrations of specific cytoplasmic glucocorticoid receptors. A similar response was observed in seven of the lines irrespective of their concentration of cytoplasmic receptors and pharmacological doses of steroid, well above those required to saturate receptors in cell-free extracts, were required for a massive lethal response. One cell line derived from Burkitt's lymphoma was refractory to lethal effects even with pharmacological doses of steroid. A similar unresponsiveness to the cytolethal effect of prednisolone in vitro was observed in fresh lymphoblasts derived from patients with acute lymphoblastic leukaemia despite evidence of a satisfactory clinical response to therapy which included steroid. The resistance of human lymphoblastoid cells to treatment with glucocorticoids in vitro may result from a defect in activation subsequent to the binding of steroid to cytoplasmic receptors.     

Failure of human myeloma cells to heterotransplant into the brain of athymic rats

Sixty-four separate intracranial inoculations of bone marrow cells obtained from 26 patients with human myeloma were performed and the animals were kept under observation for 9-10 months. Samples were obtained from a heterogenous group of patients with diverse types of paraprotein production, clinical status, and response to treatment. Inocula size ranged from 3.5 x 10(5) nucleated cells to about 2 x 10(7), while the percentage of plasma cells varied from nondetectable to 90%. Only one animal (of 2) injected with an aliquot of the bone marrow aspirate from a patient developed a small, clinically undetectable tumor, noticed at the end of the observation period. No other animal developed tumors. Thus, our studies indicate that the intracerebral inoculation of human myeloma cells may not be a profitable means of establishing additional human myeloma cell lines.