Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38 MPC-1- CD49e- immature, CD38 MPC-1+ CD49e- intermediate and CD38 MPC-1+ CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1- CD45+ CD49e- but not MPC-1- CD45- CD49e- immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1- CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a non-tumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Recently, there has been an increasing interest in the expression pattern and biological significance of the CD45 molecule in myeloma cells. In this study, we have further defined the phenotypic pattern of CD45 expression on myeloma cells. Using a panel of myeloma cell lines, we showed that CD45 showed a remarkably heterogeneous pattern of expression. Whereas some cell lines were CD451 and others were CD452, the U-266 cell line, although predominantly CD452, still had a considerable subpopulation of CD451 cells. Among the myeloma cell lines examined, there was a direct correlation between interleukin-6 (IL-6) dependency and CD45 positivity. Moreover, we showed that IL-6 stimulation led to the induction of expression of CD45 and cellular proliferation. Using independent experimental approaches, we could show that the IL-6–induced effects were closely linked to CD45 expression. First, sorting out CD451 and CD452 subsets of U-266 cell line followed by IL-6 stimulation, only the CD451 cells showed a proliferative advantage after IL-6 stimulation. Second, IL-6 stimulation of sorted CD452 cells was gradually followed by phenotypic conversion to CD451 cells that started after 2 days as judged by the detection of CD45 mRNA by reverse transcription polymerase chain reaction (RT-PCR) and immunophenotypic analysis by flow cytometry. Withdrawal of IL-6 from the medium led to gradual loss of CD45 expression in CD451 flow-sorted U-266 cells. Third, the use of vanadate, a potent inhibitor of protein tyrosine phosphatase (PTP), abrogated the IL-6–induced proliferation in the CD451 myeloma cells. On the other hand, cellular proliferation induced by IL-6 was not affected by the serine-threonine phosphatase inhibitor okadaic acid. Our data show that the expression pattern of CD45 in myeloma cell lines is heterogeneous and show for the first time that CD45 expression can be induced by IL-6 stimulation. Finally, these data shed some light on the biological role of CD45 in myeloma by determining the proliferative population among myeloma cells.

The expression of Pax-5 gene is altered in human myeloma cells (malignant plasma cells). This altered expression is considered to be closely involved in oncogenesis of human myeloma. To investigate the possible mechanism(s) underlying this alteration, we first cloned the 1,050 bp fragment in the 5’ upstream region of human Pax-5 gene by PCR-mediated gene walking method. The cloned fragment has predicted regulatory motifs for Lyf-1(Ik-1), Ik-2, bHLH, E-47, Sox-5, Oct-1, GATA-1,-2, and -3, but it lacks a TATA box. By constructing deletion mutants of this fragment, its basal promoter activity was analyzed by transfecting these mutants to Cos 7 cells. The maximal promoter activity was recovered by the fragment that extends between -70 to -820 upstream of the transcription start site. Also, three DNA fragments from this cloned sequence were used as templates in gel shift assay; these fragments covered most of the predicted regulatory sites. Specific binding activities were found in each DNA fragment. Therefore, we could clone the functionally active fragment of 5’ upstream region of human Pax-5 gene.

Recent phenotypic analysis of plasma cells showed that normal plasma cells do express the B-cell lineage specific molecule CD19, but their malignant counterpart (myeloma cells) are CD19-. To clarify the meaning of loss of CD19 antigen on myeloma cells, we first compared the expression of CD19 and Pax-5 genes among B cells, normal plasma cells, myeloma cell lines, and primary myeloma cells, because the Pax-5 gene was reported to encode the transcriptional factor, 6-cell-specific activating protein (BSAP), necessary for CD19 gene expression. Neither CD19 nor Pax-5 mRNA could be detected in those primary myeloma cells and cell lines, whereas normal plasma cells did express both CD19 and Pax-5 mRNA. Furthermore, we could confirm that BSAP-binding activity was not detected in the nuclear extract from CD19- myeloma cell line (KMS-5) but was detected in CD19+ B-cell line (Raji by gel shift assay. We further examined the expression of E2A and Id gene, because E2A and Id are considered to be positive and negative regulators in the expression of Pax-5 gene, respectively. However, no significant differences in the expression of the E2A and Id-2 genes could be observed between myeloma cells and normal plasma cells. Therefore, these data suggest that the altered expression of Pax-5, but not E2A or Id, is responsible for the loss of CD19 expression in human myeloma cells, although the underlying mechanism of the altered Pax-5 gene expression remains to be clarified.

By using two-color phenotypic analysis with fluorescein isothiocyanate-anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) significantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM-102). or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-pl (TGF-β1) and macrophage inflammatory protein-1p (MIP-1β) could suppress survival of pre-B cells even in the presence of 11-7. Plasma cells alone could not suppress survival of pre-B cells in the presence of IL-7, but coculture of plasma cells with KM-102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF- β 1, and MIPl β mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF-p1 and MIP-lp mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated t o secrete lesser levels of supporting factor (IL-7) and higher levels of inhibitory factors (TGF- βI and MIP-1 β) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

In the peripheral blood (PB) we detected so-called early plasma cells that might already be committed to entering the bone marrow (BM). By two-colour staining with FITC-anti-CD38 antibody, their intensity (CD38++) of expression of CD38 antigen was between that of germinal centre (GC) B cells (low expression (CD38+)) and that of BM plasma cells (high expression (CD38++)), and their phenotype was CD38++ CD19+ CD10- CD20- CD21+ CD24- CD39+ CD5- VLA-4+ VLA-5- MPC-1- without expression of surface membrane IgM (SmIgM). Morphological and immunological examination of the sorted cells confirmed that they were plasmacytoid cells with expression of cytoplasmic IgG (cIgG). Variations of these early plasma cells were examined in various diseases. In active systemic lupus erythematosus, bacterial septicaemia and liver cirrhosis, early plasma cell levels were significantly increased in PB, and after subsidence of such inflammation (inactive states) these cells returned to normal levels. In contrast, normal early plasma cells were significantly suppressed in myelomas, whilst normal or slightly increased numbers of early plasma cells was found in benign monoclonal gammopathy (BMG). In addition, the number of normal early plasma cells returned to a normal level in myeloma cases with complete responses. Therefore, early plasma cells were identified phenotypically, and an increase and decrease in these cells in PB may reflect mobilization and suppression, respectively, of activated B cells into BM plasma cells.

Using two-colour phenotypic analysis with anti-CD38 antibody, human myeloma cells can be classified into VLA-5- immature and VLA-5+ mature cells. We examined the relationship between variations of these subpopulations and clinical responses during treatment in multiple myeloma (MM). 39 patients with MM were treated with combined chemotherapy. First estimation of clinical responses after induction therapy showed that early clinical responses were correlated with the percentage of immature myeloma cells present after induction therapy (P < 0.01), not at diagnosis. After three courses of cyclic maintenance therapy, immature myeloma cells significantly decreased in proportion along with a decrease in total myeloma cells in maintained or more responsive cases (P < 0.01). On the other hand, immature myeloma cells were still found in high proportions in nonresponsive cases with no change (NC) or minor response (MR) (P < 0.01). Furthermore, in relapsing cases from partial response (PR) or progressive disease (PD) from nonresponsive cases, immature myeloma cells increased markedly. Therefore these results show that high proportions of VLA-5- immature myeloma cells remaining after induction therapy and during maintenance therapy correlate well with a declining clinical course of MM during maintenance therapy.