Expression In Myeloma Cells Research Articles

Positive Regulatory Domain I Binding Factor 1 Silences Class II Transactivator Expression in Multiple Myeloma Cells

The major histocompatibility complex (MHC) class II transactivator (CIITA) acts as a master switch to activate expression of the genes required for MHC-II antigen presentation. During B-cell to plasma cell differentiation, MHC-II expression is actively silenced, but the mechanism has been unknown. In plasma cell tumors such as multiple myeloma the repression of MHC-II is associated with the loss of CIITA. We have identified that positive regulatory domain I binding factor 1 (PRDI-BF1), a transcriptional repressor, inhibits CIITA expression in multiple myeloma cell lines. Repression of CIITA depends on the DNA binding activity of PRDI-BF1 and its specific binding site in the CIITA promoter. Deletion of a histone deacetylase recruitment domain in PRDI-BF1 does not inhibit repression of CIITA nor does blocking histone deacetylase activity. This is in contrast to PRDI-BF1 repression of the c-myc promoter. Repression of CIITA requires either the N-terminal acidic and conserved PR motif or the proline-rich domain. PRDI-BF1 has been shown to be a key regulator of B-cell and macrophage differentiation. These findings now indicate that PRDI-BF1 has at least two mechanisms of repression whose function is dependent on the nature of the target promoter. Importantly, PRDI-BF1 is defined as the key molecule in silencing CIITA and thus MHC-II in multiple myeloma cells.

Ku86 variant expression and function in multiple myeloma cells is associated with increased sensitivity to DNA damage.

Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination. Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells. Tumor cells (CD38(+)CD45RA(-)) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v). Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus. EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity. Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity. Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86. Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents. Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.

Assessment of cellular expression of parathyroid hormone-related protein mRNA and protein in multiple myeloma.

The capacity of multiple myeloma cells to generate parathyroid hormone-related protein (PTHrP) has been examined by in situ assessment of PTHrP mRNA and PTHrP protein in myeloma cells of patients in whom the disease was associated with the development of hypercalcaemia. The presence of PTHrP mRNA was evaluated by in situ hybridization using an antisense riboprobe, and PTHrP by immunohistochemistry using a monoclonal antibody, in archival bone marrow trephine specimens from 17 patients. PTHrP mRNA was detected in myeloma cells in 16 of the 17 patients, indicating a high frequency of PTHrP gene expression in myeloma cells in these subjects. PTHrP protein was, on the other hand, detected in the myeloma cells of only five of these patients. The impact of the mercury-based fixation and decalcification procedure used for processing the bone marrow trephine specimens was assessed to determine the influence of this process on the outcome of the immunohistochemical assay for PTHrP. It was shown that this preparative procedure resulted in a marked reduction of immunohistochemically detectable PTHrP, which provides a possible explanation for the lower frequency of positivity for PTHrP in myeloma cells in the bone marrow specimens. The present findings are consistent with the view that PTHrP can be generated in myeloma cells in vivo, and could contribute to osteolysis and hypercalcaemia, as in patients with cancer.

Assessment of cellular expression of parathyroid hormone-related protein mRNA and protein in multiple myeloma

The capacity of multiple myeloma cells to generate parathyroid hormone-related protein (PTHrP) has been examined by in situ assessment of PTHrP mRNA and PTHrP protein in myeloma cells of patients in whom the disease was associated with the development of hypercalcaemia. The presence of PTHrP mRNA was evaluated by in situ hybridization using an antisense riboprobe, and PTHrP by immunohistochemistry using a monoclonal antibody, in archival bone marrow trephine specimens from 17 patients. PTHrP mRNA was detected in myeloma cells in 16 of the 17 patients, indicating a high frequency of PTHrP gene expression in myeloma cells in these subjects. PTHrP protein was, on the other hand, detected in the myeloma cells of only five of these patients. The impact of the mercury-based fixation and decalcification procedure used for processing the bone marrow trephine specimens was assessed to determine the influence of this process on the outcome of the immunohistochemical assay for PTHrP. It was shown that this preparative procedure resulted in a marked reduction of immunohistochemically detectable PTHrP, which provides a possible explanation for the lower frequency of positivity for PTHrP in myeloma cells in the bone marrow specimens. The present findings are consistent with the view that PTHrP can be generated in myeloma cells in vivo, and could contribute to osteolysis and hypercalcaemia, as in patients with cancer.

A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes

Major histocompatibility complex (MHC) class II molecules play a central role in immune responses, and transcription of this family of genes requires the MHC class II transactivator (CIITA). CIITA has four promoters, which are transcribed in a tissue-specific manner. CIITA promoter III is constitutively active in mature B-lymphocytes. This report now describes the minimal 319-base pair promoter region necessary for maximal transcriptional activity in B-lymphocytes. Ultraviolet light and dimethylsulfate in vivo genomic footprinting analyses reveal five occupied DNA sequence elements present in intact B-lymphocytes. Functional analysis of these elements using promoter deletions and site-specific mutations demonstrates that at least two of the sites occupied in vivo are critical for transcriptional activity. In vitro protein/DNA analysis suggests that one of the sites is a TEF-2-like element and the other is occupied by a novel transcription activator. In addition, nuclear factor-1 associates with the promoter both in vivo and in vitro. In myeloma cell lines, loss of CIITA transcription correlates with a completely unoccupied CIITA promoter III. These findings suggest that CIITA transcription in B-lymphocytes is activated through at least two strong promoter elements, while loss of expression in myeloma cells is mediated through changes in promoter assembly.

Potential role of insulin-like growth factor binding protein-4 in the uncoupling of bone turnover in multiple myeloma.

Decreased bone formation plays an important role in the development of lytic lesions during the late stage of multiple myeloma (MM). Release of insulin-like growth factor binding protein-4 (IGFBP4) by tumour cells adjacent to bone may inhibit IGF-I-stimulated osteoblast growth and contribute to decreased bone formation. The present study demonstrates that the human MM cell line, ARH-77, expresses IGFBP4 and, to a lesser extent, IGFBP6 mRNA and protein. IGFBP4 expression in myeloma cells may be modulated by cytokines released by stromal cells and T cells in the microenvironment. We tested the effect of recombinant interferon-gamma (INF) on IGFBP4 expression in ARH-77. INF increased IGFBP4 mRNA and protein levels at 12 h, with a decline to baseline by 24 h. In contrast, IGFBP4 was not regulated in response to IL-6, TNF-alpha, PDGF BB, bFGF, TGF-beta or the cAMP agonist, forskolin. In other systems. IGFBP4 may also be regulated post-transcriptionally by a protease that is activated by IGF-I or -II. Conditioned medium from ARH-77 cultures incubated with IGF-I or -II for up to 24 h failed to demonstrate proteolytic activity. Proteolysis was also not observed when conditioned medium containing exogenous rhIGFBP4 was incubated with IGF-I or -II under cell-free conditions. To determine if human myeloma tumours also express IGFBP4, total RNA was isolated from four tumour biopsies. All samples expressed detectable levels of IGFBP4 mRNA. These findings indicate that interferon-gamma may indirectly modulate bone formation via the the release of tumour-derived IGFBP4. suggesting that the immune system may influence bone turnover in MM. Failure of myeloma cells to release protease activity may promote IGFBP4 accumulation in the microenvironment during tumour growth.

Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells.

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12-48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.

MDM2 Protein Overexpression Promotes Proliferation and Survival of Multiple Myeloma Cells

AbstractThe murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange–ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.

The Retinoblastoma Susceptibility Gene RB-1 in Multiple Myeloma

Genetic mechanisms leading to the development of multiple myeloma (MM) remain poorly understood. Given the frequency of chromosome 13 deletion in MM and the localization in 13q14 of the retinoblastoma susceptibility gene RB-I, an involvement of RB-1 in MM pathogenesis has been proposed. Moreover, interleukin-6 (IL-6) has been shown to be the main growth factor for MM in vitro and in vivo. The product of the RB-I gene (pRB) can down-regdate IL-6 gene expression. Absence of pRB may then induce an autocrine IL-6 expression in myeloma cells and contribute to the autonomous growth of MM. As assessed in this review, heterozygous deletion of RB-I is very common in MM but does not alter gene transcription and protein expression. Nevertheless, homozygous deletion of RB-1 has been identified in some h4M patients with advanced disease and in the IL-6-autocrine human myeloma cell line U266. Thus, even if inactivation of RB-I appears to be only a rare and late oncogenic event in MM and is not likely to represent the main mechanism involved in IL-6 up-regulation in MM, definitive assessement of the actual role played by RB-I in MM pathogenesis still needs further investigation particularly the examination of pRB function.

Altered Expression of Pax-5 Gene in Human Myeloma Cells

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, B-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 genes, 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 these 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.

Roles of Protein Phosphatase 1 and 2A in an IL-6-Mediated Autocrine Growth Loop of Human Myeloma Cells

Deregulation of IL-6 production is one of the major causes for human multiple myeloma. Exogenous IL-6 stimulated the proliferation of fresh human myeloma cells and the myeloma cell line, U266, which produced IL-6 spontaneously. Anti-IL-6 antibody and IL-6 antisense oligonucleotide suppressed the IL-6-stimulated myeloma cell proliferation, indicating that IL-6 induced the myeloma cell proliferation via an autocrine loop. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, inhibited the U266 cell proliferation at a concentration of less than 1 ng/ml. At this concentration, okadaic acid suppressed the IL-6-induced IL-6 gene expression of myeloma cells. It seems that the okadaic acid blocked the myeloma cell proliferation by reducing IL-6 synthesis in myeloma cells. In addition, IL-6 itself also regulated IL-6 receptor expression. Analysis by FACScan and RT–PCR showed that anti-IL-6 antibody treatment up-regulated IL-6 receptor expression. Interestingly, the presence of okadaic acid induced the up-regulation of IL-6 receptor expression as well as the down-regulation of IL-6-induced gp130 phosphorylation in the myeloma cells. Taken together, these data suggest that protein phosphatase 1 and 2A are involved in IL-6-mediated autocrine growth of human myeloma cells by modulating IL-6 signaling and IL-6 receptor expression in myeloma cells.

Interleukin-6 gene expression is preferentially restricted in VLA-5-MPC-1- immature but not in VLA-5+MPC-1+ mature myeloma cells.

Interleukin-6 (IL-6) is essential for the proliferation of myeloma cells, and IL-6 is considered to be produced from not only myeloma cells themselves but also microenvironments including stromal cells. To clarify which subpopulation of myeloma cells can produce IL-6, we examined IL-6 mRNA expression in immature and mature myeloma cells and normal plasma cells by RT-PCR. IL-6 mRNA expression was found in all (10/10) specimens of sorted VLA-5-MPC-1- immature myeloma cells and 27% (3/11) of VLA-5-MPC-1+ myeloma cells. On the contrary, no IL-6 mRNA was expressed in VLA-5+MPC-1+ mature myeloma cells (0/4) and CD19+CD56- normal plasma cells (0/5). IL-6R gene expression was detected in all normal and malignant plasma cells without exception. Therefore, these findings suggest that IL-6 production is preferentially restricted in immature not mature myeloma cells, and this may explain why immature myeloma cells show greater proliferative activity.

Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells

(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.

Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.

CD40 expression in malignant plasma cells. Role in stimulation of autocrine IL-6 secretion by a human myeloma cell line.

Myeloma is a neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. In these studies, we have demonstrated that CD40 is expressed in human myeloma cells and have used a recently established IL-6-dependent myeloma cell line, ANBL-6, to examine the potential function of CD40 expression in myeloma cells. In addition to its expression on the ANBL-6 cells, we show that CD40 is expressed on freshly isolated myeloma cells from seven of seven patients tested. To address the role of CD40 expression in myeloma cells, we have examined the responsiveness of the ANBL-6 cell line to a CD40-specific mAb, G28-5. This cell line has previously been shown to proliferate only in response to IL-6. Of interest in this study, G28-5 also induced proliferation of the ANBL-6 cells. This proliferation was substantially inhibited by an IL-6-neutralizing mAb. Analysis of ANBL-6 cell culture supernatants by ELISA demonstrated that G28-5-stimulated cells secreted significant levels of IL-6, whereas unstimulated cell culture supernatants contained undetectable levels of IL-6. Furthermore, CV-1/EBNA cells expressing the human CD40 ligand also induced the proliferation of the ANBL-6 cell line, an effect that was inhibited by the anti-IL-6 mAb. Lastly, RNA blot analysis demonstrated an increase in IL-6 message in G28-5-stimulated ANBL-6 cells over unstimulated cells. These results indicate that the primary mechanism of anti-CD40-stimulated proliferation of the ANBL-6 cells is the induction of autocrine IL-6 production. Moreover, these data suggest that the expression of CD40 in malignant plasma cells may play a role in tumor cell expansion, possibly by stimulating the induction of autocrine IL-6 secretion.

Differential regulation of interleukin‐6 receptors by interleukin‐6 and interferons in multiple myeloma cell lines

Interleukin-6 (IL-6) mediates pleiotropic functions through specific receptors (IL-6R) composed of an 80-kDa binding protein, associated with a non-ligand binding protein (gp130) which transduces the signal. Because IL-6 is the major tumor growth factor in multiple myeloma, we investigated the regulation of IL-6R in two human multiple myeloma cell lines. Binding experiments with 125I-labeled IL-6 showed that IL-6R were expressed at a high density on RPMI-8226 cells (15 000 receptors/cell), but no specific binding was detected on XG-1 cells, whose growth depends on the presence of exogenous IL-6. However, when IL-6 was removed from the culture medium, high-affinity IL-6R appeared on the surface of XG-1 cells (5300 sites/cell). Treatment of RPMI-8226 cells with IL-6 reduced the number of IL-6R without changing their affinity. This reduction was dose dependent and was not affected by acid treatment which dissociates ligand-receptor complexes. Cross-linking experiments showed that the formation of one IL-6/receptor complex of 160 kDa markedly decreased upon IL-6 treatment, while the other complex of 190 kDa became undetectable. These data provide evidence for ligand-induced down-regulation of membrane IL-6R expression in myeloma cells. Treatment of RPMI-8226 cells with interferon-alpha (IFN-alpha), which inhibits the growth of these cells, stimulated IL-6R expression and increased the formation of the 160-kDa IL-6/receptor complex. This stimulation was specific for IFN-alpha, since IFN-gamma reduced the number of IL-6R. These data indicate that, in myeloma cells, IL-6R are differentially regulated by IL-6 and IFN-alpha.

High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells

We show that the mouse γ2b heavy chain or human β-globin 3′ untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse γ2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3′ untranslated sequences. Cell lines transfected with these constructs, pSVtPA(tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 μg/ml) in comparison with the parental 59D8 myeloma cell line (7.6–10 μg/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(β), in which the 3′ untranslated region of the mouse γ2b heavy chain or human β-globin gene was substituted for the 3′ untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).

Expression of Fibronectin and Adhesion to Fibronectin in Myeloma Cell Lines

Fibronectin (FN) expression in six myeloma, two mature B-cell lines, and four T-cell lines was analyzed. All myeloma cell lines expressed FN at various levels, while mature B- and T-cell lines apparently had less FN. Moreover, an extramedullary plasmacytoma-derived myeloma cell line, KHM7, was found to secrete FN into the culture medium. Fibronectin receptors, VLA4 or VLA5, were expressed at various levels on all myeloma cell lines. An adhesion assay revealed three of six myeloma cell lines bound to FN. However, there was no correlation between binding to FN and FN receptor expression, indicating a complicated FN binding pathway. The mechanism and pathological significance of FN expression and FN binding in myeloma cells are discussed.

Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system.

We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles.

多発性骨髄腫とＩＬ‐６

Myeloma cells produce IL-6 constitutively, and express its receptor on their surfaces. In vitro, exogenously added IL-6 promotes the growth of myeloma cells from about half of the patients. Therefore, IL-6 is considered as an autocrine growth factor for some myeloma cells. Plasma cells obtained from bone marrow aspirates of benign monoclonal gammopathy (BMG) cases produced IL-6 transiently only after IL-1 stimulation, but not constitutively. Therefore, constitutive production of IL-6 is considered to be characteristic of transformed plasma cells and involved in pathogenesis of multiple myeloma. In two cases with cytokine producig tumors, IL-6 producing renal cell carcinoma and IL-1 producing lung cancer, we could confirm the biological activities of IL-1 and IL-6 on myeloma cells and plasma cells in vivo. To understand the mechanism of the constitutive expression of IL-6 gene in myeloma cells, first, we performed Southern blotting analysis, but we found no gross structural abnormality of the IL-6 gene in myeloma cells. Second, we studied NF-IL6 mRNA expression in myeloma cells by RT-PCR method; NF-IL6 is a nuclear factor that specifically bind the promoter region of the IL-6 gene and activate its transcription. NF-IL6 mRNA expression was detected in a human myeloma cell line, KMS-5 and freshly isolated myeloma cells. Therefore, NF-IL6 may be involved in constitutive IL-6 production in myeloma cells.