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Cell lines RPMI 8226, JJN3, U266 B1, NCI-H929 (all EBV−) and ARH77 and HS-Sultan (both EBV+) have been extensively characterized in this study. EBV− lines expressed the phenotype (CD138−, CD19+, CD20+) whereas EBV+ were (CD138+, CD19−, CD20−). CD56 expression was restricted to EBV− cell lines, with the exception of U266 B1, whereas PCA-1 was strongly expressed on five of the six cell lines. Only EBV+ cell lines bound peanut-agglutinin (PNA). However, all cell lines bound the lectin Jacalin that binds the same receptor as PNA, irrespective of the receptors sialylation status. By RT-PCR and direct sequencing of their IgH V/D/J domains, ARH77 was demonstrated to use the germline sequence VH4-34/dm1/JH6b, whereas no arrangement was demonstrated for RPMI 8226, suggesting IgH gene deletion or mutation. HLA class I and II antigens were detected using HLA typing on all cell lines warranting their use as suitable targets for HLA-restricted cytotoxic T cells. By sensitive RT-PCR, mRNA for IL-6, IL-6R and TNFβ was found expressed in all cell lines. IL-1 mRNA expression was predominantly associated with the EBV+ phenotype. Although mRNA for IL-3 and GM-CSF was never detected, transcripts for c-kit ligand and, more commonly, its receptor were. Likewise GM-CSF, M-CSF and erythropoietin mRNA transcripts were detected in the majority of cell lines.
The phenotypic profile of malignant plasma cells is variable both between and within individual samples from patients with multiple myeloma (MM) (Kawano et al, 1995; San Miguel et al, 1995). Consequently, myeloma plasma cell isolation for in vitro study remains problematic. Because human plasma cell-like cell lines are assumed to reproduce at least some of the characteristics of primary myeloma cells, they represent a readily available alternative to the potentially heterogenous primary myeloma cell population.
Some doubt persists however, as to the eventual usefulness of using cell lines in decoding the signalling events between myeloma cells and the non-tumour cells of the bone marrow. Additional molecular events must occur before primary myeloma cells can be cultured indefinitely in vitro (Zhang et al, 1994; Shimizu et al, 1992). Cell lines are de-differentiated outgrowths of terminally differentiated mature myeloma plasma cells, with a subsequent morphological resemblance to lymphoblastoid cells and are potentially unrepresentative of myeloma cells in any stage of the disease. In addition, some myeloma cells are known to contain EBV nucleotide sequences. Further characterization of the original tumour is not possible, but without this it is uncertain as to whether the EBV transformed cells actually originate from the malignant clone (Ralph, 1985; Drewinko et al, 1985; Pellat-Deceunynk et al, 1995).
Myeloma cell lines have been a more valuable and reliable resource where direct conclusions relating to MM per se have not been sought. For example, investigations into the regulation of immunoglobulin (Ig) gene, the signalling requirements of differentiating germinal centre B cells and the mechanisms of post-transitional modifications of proteins. Furthermore, myeloma cell lines have been used as targets for anti-tumour chemotherapy, immunotoxins, oligonucleotide anti-sense strategies, and for investigating drug resistance. Finally, myeloma cell lines have a use in the development of human hybridoma systems.
Although myeloma cell lines are not representative of primary myeloma cells, their sharing of fundamental characteristics, i.e. Ig gene re-arrangement, cytoplasmic idiotype expression and the production of, or requirement for, a number of peptide regulators, e.g. IL-6, make them ideal for use in the development and optimization of therapeutic strategies such as graft purging and the semi-quantitative molecular monitoring of clonal tumour cells in patients after high-dose therapy. Furthermore, myeloma cell lines may have a use as model HLA-matched targets for cytotoxic T cells generated in vitro. This study therefore describes the phenotypic and molecular characterization of a number of readily available cell lines obtained from patients with plasma cell dyscrasia.
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Human cell lines sharing characteristics with B-lineage cells obtained from patients with plasma cell dyscrasia may act as useful in vitro models for the study of multiple myeloma. To this end, other investigators have stressed the importance of cell surface phenotype in differentiating plasma cell-like cell lines from EBV-positive cell lines (Pellat-Deceunynk et al, 1995). In this study a clear distinction could be made between the EBV-negative lines RPMI 8226, JJN3, U266 Bl, NCI-H929 and the EBV-positive HS-Sultan and ARH77 simply on the basis of syndecan-1 (CD138) expression on the former and CD19 expression on the latter. Investigation of other B-cell-specific antigens (CD10 and CD20), surface light chain expression, and plasma cell antigens (PCA-1, CD38 and CD56) confirmed two phenotypically distinct cell types (Table IV).
The N-CAM CD56 was previously found on HS-Sultan (37%) and ARH77 (13%) (Hiroshi et al, 1992), but in our study was not detected on either cell line. PCA-1 has been found expressed on primary plasma cells from pleural effusions or peripheral blood from patients with myeloma, suggesting its expression is related to malignant cell escape from the bone marrow into the circulation (Hata et al, 1994).
These phenotypic data generally corroborate previous investigations, although differences in the level of specific staining may have resulted from the use of different antibodies and changes in cell lines with time as previously found (Kozbor et al, 1983; Barut et al, 1992). A failure to detect Ig in any cell line conditioned medium may reflect compromised Ig production, assembly and secretion. IgH gene deletion has probably occurred in RPMI 8226.
PNA binds to primary bone marrow plasma cells (Rhodes & Flynn, 1988; Slupsky et al, 1992), but three of the four EBV-negative lines did not bind PNA. Both EBV-positive lines bound PNA, a not unexpected finding as PNA binds to EBV transformed Burkitt's lymphoma cell lines (Galili et al, 1981). CD44 is a major cell surface ligand for PNA and its glycosylation may modulate this interaction (Slupsky et al, 1992). The incompletely sialylated form of CD44 binds PNA, whereas completely sialylated CD44 does not. Culture conditions may effect the sialylation profile of the cell lines thus altering PNA binding. HS Sultan, ARH77 and RPMI 8226 have been reported to express CD44 (43%, 97% and 98% positive cells respectively) (Hiroshi et al, 1992). We failed to detect CD44 expression on HS Sultan, plus decreased expression by RPMI 8226, suggesting either loss of CD44 expression or masking of the antibody binding epitope. The latter suggestion is more likely as all cell lines bound Jacalin, a lectin binding to the same structure as PNA but which is not influenced by adjacent sialyic acid residues.
We have successfully identified the VH family and VH gene used by five of the six cell lines. ARH77 has a rearranged VH 4–34 gene. The VH4–34 gene has rarely if ever been found to be associated with MM (Rettig et al, 1996), but is mandatory for the production of pathological Ig responsible for anti-erythrocyte autoimmune conditions such as cold haemagglutinin disease (Chapman et al, 1996).
In myeloma and MGUS naturally occurring idiotypic immunity has been described (Yi et al, 1995). Therapies using idiotype vaccination in the presence of growth factors to encourage HLA restricted T-cell cytotoxicity have been proposed (Österborg et al, 1998). Data presented here allow a rational approach to the investigation of idiotypic antigen presentation in the context of class I MHC molecules. HLA peptide motif search results (data not shown) demonstrated that the Ig coded for by the cell lines JJN3 and NCI-H929 did not contain peptide motifs which would associate with the HLA class I molecules expressed by these cell lines. Although not unexpected, these findings should be treated with caution as the software used to perform the HLA peptide motif search is largely based upon empirical data from conventional peptide elution studies and the theoretical likelihood of particular sequences binding to certain HLA molecules. Thus, findings obtained using this software should be corroborated using conventional methods, a task outside the scope of the present work. Regardless of these limitations, peptide motif search results identified the following peptide ALGQGLEWV from U266 Bl to have a high likelihood of association with HLA-A2, but this sequence was within the framework 2 region. This cell line sequence differs from that observed in the germline sequence 1-02 by a proline instead of leucine, at position 2 of the peptide. A comparable analysis of the entire germline Ig sequence of 1-02 failed to show any likely association of peptide motif with HLA-A2.
IL-6 is regarded as the prototype myeloma cell growth factor primarily because IL-6 knockout mice are incapable of developing plasma cell tumours (Hilbert et al, 1995). Barut et al (1992) previously reported the lack of IL-6 mRNA and protein expression by RPMI 8226 and HS Sultan, and no evidence of IL-6R mRNA or protein expression by ARH77 and HS Sultan. JJN3 was reported to be IL6 dependent, secreting low levels of IL-6 protein (Hamilton et al, 1990). Both U266 Bl and RPMI 8226 express the IL6-R (Lemoli et al, 1994) although controversy exists as to whether or not they produce IL-6 and are IL-6 dependent (Taetle et al, 1994; Barut et al, 1992; Schwab et al, 1991; Diamant et al, 1996). In this study IL-6 and IL-6R mRNA transcripts were detected in all cell lines. These discrepancies may be due to differences in sensitivity between the different detection methods or changes in cytokine production in culture with time.
Production of IL-1 may be of pathological significance in the biology of multiple myeloma mainly because of its ability to act as an osteoclast-activating factor and influence IL-6 production via PGE2. Healthy individuals express the gene for IL-1β (but not IL-1α) constitutively in their peripheral blood and bone (Cluitmans et al, 1995), but in patients with MM, the source of IL-1 has been controversial (Lichtenstein et al, 1989; Cozzolino et al, 1989; Borset et al, 1993). Recently Costes et al (1998) demonstrated IL-1β protein production in tumoural samples of the majority of patients with MM. In situ hybridization and immunohistochemistry defined myeloid and megakaryocytic cells as the major IL-1β producing cells. Production by MM plasma cells was at a significantly lower level. Furthermore, medullary but not circulating tumoural cells expressed the IL-1β gene and no IL-1β transcript was found in a panel of nine myeloma cell lines which included JJN3, RPMI 8226 and U266. Here, all EBV-negative cell lines were negative for IL-1 transcripts using 31 cycles of PCR. Increased cycle number enabled the detection of IL-1α and IL-1β transcript in RPMI 8226 and JJN3 respectively.
GM-CSF, M-CSF and erythropoietin, but not IL-3 or G-CSF mRNA transcripts, were expressed by the cell lines. Stem cell factor (c-kit ligand) transcript was detected in ARH77 and JJN3 alone, whereas the transcript for its receptor (c-kit) was detected in the majority of the cell lines. Recent studies have reported absence of c-kit on normal plasma cells and its presence on approximately one third of fresh myeloma plasma cells where binding to c-kit ligand is able to stimulate plasma cell growth (Lemoli et al, 1994; Ocqueteau et al, 1996). Absence of c-kit ligand mRNA in U266 Bl, and presence in RPMI 8226 is confirmed here (Lemoli et al, 1994).
We report for the first time that the majority of the putative myeloma cell lines derived from patients with plasma cell dyscrasia express erythropoietin mRNA. Expression of erythropoietin receptors on a human myeloma cell line has been reported previously (Okuno et al, 1990), and their expression on myeloma cells in vivo has been linked to disease progression to plasma cell leukaemia in a patient who received erythropoietin therapy (Olujohngbe et al, 1997). Our additional finding that some of the cell lines also express an erythropoietin mRNA transcript variant at low level is also novel, and further work is warranted to understand the significance of this finding.