B-lymphoid Cell Lines Research Articles

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP.

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

Binding receptors for alpha-L-fucosidase in human B-lymphoid cell lines.

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of alpha-L- fucosidase in lymphoid cells. The binding of alpha-L-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding alpha-L-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.

BCR/ABL enhances growth of multipotent progenitor cells but does not block their differentiation potential in vitro.

We have developed an in vitro system for investigating the direct effect of the BCR/ABL oncogene on the growth and differentiation of single multipotent progenitor cells (MPPC). Using this system we demonstrate that MPPC expressing P210 BCR/ABL have an enhanced ability to grow in the presence of very low concentrations of IL-3 or steel locus factor (SLF). MPPC’s expressing P210 BCR/ABL do not exhibit a block in differentiation and can give rise to mast cell, macrophage, granulocyte and B-lymphoid cell lines. Differentiation of the B-lymphoid cell lines can progress to the pre B-cell stage, marked by expression of CD45R (B220) and rearrangements of both heavy chain immunoglobulin gene alleles. Transformation of MPPC’s, initiated by BCR/ABL progresses through defined stages that can be monitored by obvious changes in growth characteristics. These studies demonstrate that BCR/ABL may enhance the growth of MPPC’s without abrogating their differentiation potential. This system should prove useful in evaluating the effect of specific genetic events on leukemogenesis and identifying the molecular signals which regulate normal hematopoietic development.

A DR7 SPECIFIC MONOCLONAL ANTIBODY TAL13.1, RAISED AGAINST A TRANSFECTANT DETECTS IL‐4 UPREGULATED ANTIGEN ON PERIPHERAL B‐LYMPHOCYTES

A monoclonal antibody TAL13.1 was raised against mouse L cells transfected with the human HLA-DRB1*0701 gene. This antibody was found to be polymorphic recognizing a determinant expressed by the DR7, DRB1*0701 and DRB1*0702 gene products. Four polymorphic sites unique to this specificity have been identified within the DR beta 1 domain. These are residues 11-14, 25, 30 and 71-74, one or a combination of which is postulated as being responsible for conferring the specificity of the antibody. In Western blot analysis TAL13.1 was found to react with the DR alpha beta dimer, but not with the free alpha or beta chains. However, in flow cytometry it failed to bind a DR alpha/DQ beta mixed pair transfectant confirming that it recognizes an epitope on the DR beta not the DR alpha chain. Although TAL13.1, a low affinity antibody is negative or only weakly positive on resting peripheral blood lymphocytes (PBLs), we have demonstrated that by interleukin-4 (IL-4) stimulation we can up-regulate the levels of antigen already present and gain a level of binding comparable to that found on B lymphoid cell lines (B-LCLs) where it has been found to be a valuable reagent in their characterization.

Two Isoforms of Murine hck, Generated by Utilization of Alternative Translational Initiation Codons, Exhibit Different Patterns of Subcellular Localization

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Homotypic aggregation of human cell lines by HLA class II‐, class Ia‐ and HLA‐G‐specific monoclonal antibodies

Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN, but not the class I-negative parental line, 221, showed homotypic aggregation in response to an HLA-G specific mAb (87G) and a broad reacting class I-specific mAb (IOT2). Both cell lines responded with aggregation to anti-class II mAb (TU35). The anti-class I mAb, W6/32, had no effect on all cell lines tested and two anti-beta 2-microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature-sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase inhibitors (genistein, herbimycin A). Serine/threonine protein kinase inhibitors (staurosporin, H7) partly inhibited the aggregation responses. There was no strict correlation between induction of aggregation and epitope density. FcR were not involved in the aggregation response, since F(ab')2 fragments of anti-DR mAb, L243, were as effective as the whole antibody. The aggregation was not influenced by mAb against accessory molecules previously shown to be involved directly or indirectly in homotypic aggregation [CD11a (LFA-1)/CD18/CD54 (ICAM-1), CD58 (LFA-3)/CD2, BB1/CD28, CD43, and CD44]. In conclusion, these data provide further evidence that HLA molecules are implicated in a novel, cellular aggregation phenomenon involving the cytoskeleton.

Coordination of Immunoglobulin DJH Transcription and D-to-JH Rearrangement by Promoter-Enhancer Approximation

The genes that encode the variable regions of immunoglobulin (Ig) heavy chains are encoded by three DNA segments: VH, D, and JH. During B-cell development these segments are brought together by a pair of site-specific DNA rearrangements. The first of these joins a D segment to a JH segment; the second brings a VH segment in apposition to a DJH unit. B-cell precursors that have undergone D-to-JH joining express transcripts that initiate at the 5' flanks of rearranged D segments (DJH transcription). In this study we have examined the coordination of D-to-JH rearrangement and DJH transcription. The B-lymphoid progenitor cell line HAFTL-1 cell clone, joining of distal D segments (DSP2 and DFL16) to JH is accompanied by an increase in the steady-state level of transcripts initiating 5' of the D coding region. Steady-state transcription of a DSP2 gene segment was undetectable prior to rearrangement and was observed to increase at least 20-fold upon joining to JH. In contrast, transcription from the 5' flank of DQ52, which lies within 700 bp of the JH cluster, was detected prior to rearrangement and did not increase significantly after rearrangement. The 5' flank of a DSP2 segment was found to support expression of a heterologous gene upon transfection into B progenitor cell lines. Expression from this DSP2 promoter was at least 30-fold higher in the presence of the Ig heavy-chain enhancer, in either orientation, than in its absence. A DNA fragment spanning the interval from -165 to +19 bp relative to the major DSP2 transcriptional start site retained enhancer-dependent promoter activity. These observations imply that activation of DSP12JH and DFL16JH transcription is coordinated with D-to-JH rearrangement by approximation of enhancer-dependent D promoter elements to the Ig heavy-chain enhancer. This interpretation is consistent with our observation that the DQ52 segment, which is closely linked to the JH cluster, is transcribed both before and after rearrangement.

Coordination of immunoglobulin DJH transcription and D-to-JH rearrangement by promoter-enhancer approximation.

The genes that encode the variable regions of immunoglobulin (Ig) heavy chains are encoded by three DNA segments: VH, D, and JH. During B-cell development these segments are brought together by a pair of site-specific DNA rearrangements. The first of these joins a D segment to a JH segment; the second brings a VH segment in apposition to a DJH unit. B-cell precursors that have undergone D-to-JH joining express transcripts that initiate at the 5' flanks of rearranged D segments (DJH transcription). In this study we have examined the coordination of D-to-JH rearrangement and DJH transcription. The B-lymphoid progenitor cell line HAFTL-1 cell clone, joining of distal D segments (DSP2 and DFL16) to JH is accompanied by an increase in the steady-state level of transcripts initiating 5' of the D coding region. Steady-state transcription of a DSP2 gene segment was undetectable prior to rearrangement and was observed to increase at least 20-fold upon joining to JH. In contrast, transcription from the 5' flank of DQ52, which lies within 700 bp of the JH cluster, was detected prior to rearrangement and did not increase significantly after rearrangement. The 5' flank of a DSP2 segment was found to support expression of a heterologous gene upon transfection into B progenitor cell lines. Expression from this DSP2 promoter was at least 30-fold higher in the presence of the Ig heavy-chain enhancer, in either orientation, than in its absence. A DNA fragment spanning the interval from -165 to +19 bp relative to the major DSP2 transcriptional start site retained enhancer-dependent promoter activity. These observations imply that activation of DSP12JH and DFL16JH transcription is coordinated with D-to-JH rearrangement by approximation of enhancer-dependent D promoter elements to the Ig heavy-chain enhancer. This interpretation is consistent with our observation that the DQ52 segment, which is closely linked to the JH cluster, is transcribed both before and after rearrangement.

Comparison of antineoplastic activity of 2',2'-difluorodeoxycytidine and cytosine arabinoside against human myeloid and lymphoid leukemic cells

2',2'-difluorodeoxycytidine (known as dFdC, Gemcitabine and LY188011) is a new analog of deoxycytidine which has demonstrated excellent antineoplastic activity against many kinds of solid tumors and leukemic cell lines. We were interested in the comparison of the antineoplastic activity of this new antimetabolite with cytosine arabinoside (ARA-C) against HL-60 myeloid, RPMI-8392 B-lymphoid and Molt-3 T-lymphoid leukemic cell lines. Our in vitro experiments showed that dFdC was a more potent cytostatic drug than ARA-C against all the leukemic lines with IC50 ranging from 3 to 10 nM for dFdC and from 26 to 52 nM for ARA-C for a 48 h exposure. The cytotoxicity of both drugs was evaluated by clonogenic assay and dFdC was found to be 100 times more potent than ARA-C against all the leukemic cell lines for both a 2 h and a 24 h exposure. The recovery of DNA synthesis after drug removal was much slower for dFdC than for ARA-C. However, in contrast to cytostatic and cytotoxicity results ARA-C was a more potent inhibitor of DNA synthesis than dFdC for all the leukemic cell lines for short exposure. Uptake and elimination of the drugs showed that dFdC accumulated to a higher degree in the leukemic cells than ARA-C and that elimination of this difluoro analog was slower than that of ARA-C. These results indicate that dFdC has more potent in vitro antileukemic activity than ARA-C.

METHYLATION PATTERN OF THE HLA‐DRα GENE IN HUMAN TISSUES

The CCGG and GCGC sites of the human HLA-DR alpha gene are hypermethylated in human tissues (including B-lymphocytes, T-lymphocytes, muscle, brain, sperm, skin, kidney, suprarenal and mammary glands) and three B-lymphoid cell lines. Therefore, the HLA-DR alpha gene can be transcribed even though extensively methylated. The only exception to the hypermethylated state of the HLA-DR alpha gene is represented by one or both of the two HhaI sites (H1 and H2) localized in the 5' portion of the gene. Analysis of the computer-generated secondary structure of the HLA-DR alpha mRNA suggests that the H1 and H2 sites belong to a region (5'-GAGCGCCCA-3'/5'-UGAGCGCUC-3') exhibiting extensive base pairing. Therefore, unmethylation of these CG sites can contribute in preventing mCG----TG/CA changes in this region, which would lead to extensive alterations of the secondary structure of the 5' portion of the HLA-DR alpha MRNA. On the other hand, the selective pressure to maintain unaltered the methylated CG dinucleotides in the coding regions of the HLA-DR alpha gene could be due to codon restrictions, since the majority of the methylation-related CG----TG or CG----CA variations would generate aminoacid changes. Accordingly, the analysis of different HLA-DR alpha genomic sequences indicates that variations of the CpG dinucleotides occur only in the non-coding portions of the HLA-DR alpha gene.

An Epstein-Barr virus immortalization associated gene segment interferes specifically with the IFN-induced anti-proliferative response in human B-lymphoid cell lines.

Immortalization of human B-lymphocytes by Epstein-Barr virus (EBV) is associated with a decreased anti-proliferative response to interferon (IFN). In the present investigation we show that the resistance to the anti-proliferative effect of IFN class I on certain EBV-carrying Burkitt lymphoma cell lines is connected to the presence of the EBNA-2 gene and parts of the EBNA-5 gene of the EBV genome. Transfection of the genomic segment comprising these open reading frames into an IFN-sensitive lymphoma cell line demonstrated that it is sufficient to make cells resistant towards the antiproliferative effect of IFN class I. Expression of the EBNA-2 gene seems to be correlated with the IFN-resistant phenotype. The antiviral function of IFN, as tested by inhibition by vesicular stomatitis virus (VSV) infection, and the IFN-receptor binding are not suppressed. The present results suggest that the neutralization of the anti-proliferative effect of IFN-alpha is involved in the EBV-mediated immortalization of B-cells and that the anti-proliferative action of IFN class I does not necessarily recruit the same mechanism as the antiviral effect.

Relative rates of RNA synthesis across the genome of Epstein-Barr virus are highest near oriP and oriLyt

The rates of Epstein-Barr virus transcription were measured in isolated nuclei from marmoset and human lymphoblasts transformed in vitro. In B95-8, a marmoset B-lymphoid cell line, the most frequently transcribed viral genes are the EBERs (small nuclear RNAs) and BHLF-1 (encoding a lytic-phase gene product). The EBERs and BHLF-1 genes are separated by nearly 50 kilobase pairs on the Epstein-Barr virus genome and lie adjacent to (less than 300 base pairs from) oriP and oriLyt, respectively. oriP and oriLyt are putative origins of viral DNA replication, and each is associated with a transcriptional enhancer element. Among the human B-lymphoblastoid cell lines tested, only the transcription of EBERs predominates.

Evidence for different interleukin 1 receptors in murine B- and T-cell lines.

Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This finding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number of IL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.

Inhibition of 'in vitro' tumor cell growth by aromatic polyamidines exhibiting antiproteinase activity.

Aromatic polyamidines containing two, three or four benzamidine residues inhibit proteinase activity and proliferation of different human tumor cell lines, including leukemic (K562, HEL), melanoma (Colo 38) and B-lymphoid (WI-L2) cell lines. In addition, the benzamidine derivatives analysed in the present study inhibit cell growth of the Chinese hamster FHO6T1-1 cell line, obtained after transfection of primary lung cells with the activated human T24-Ha-ras-1 oncogene. After treatment of FHO6T1-1 cells with benzamidine derivatives, a sharp decrease of the content of Ha-ras-1 mRNA was found, but not of transferrin receptor mRNA. We found that inhibition of cell proliferation by tetra-benzamidine derivatives is not restricted to tumor cells, but concerns also non-tumorigenic cell lines as well as normal primary fibroblasts. Therefore, our analysis was extended to di- and tri-benzamidine derivatives, which could be proposed as useful substrates in the synthesis of drug-conjugated monoclonal antibodies or growth factors. The data obtained demonstrate that these latter compounds and their halo-derivatives also exhibit strong antiproliferative effects on in vitro cultured cells.

CAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1.

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.

High frequency of plasminogen activator secretion by malignant human lymphoid cell lines of T-cell type origin.

Diffuse defibrination is rarely observed in acute lymphoblastic leukemia (ALL). Clinical and immunologic data suggest that it is more likely to occur in T cell derived ALL. The current investigation involved the secretion of plasminogen activators (PA) of tissue type (t-PA) or urokinase type (U-PA) by testing supernatants of 21 permanent human leukemic cell lines originating from various hematopoietic lineages and one induced lymphoblastoid cell line. (LCL) The amount of PA in each supernatant was determined by biologic and immunoenzymologic assays. The correspondence with the expected molecular weight (MW) according to the PA type was checked by zymography. PA secretion of U-PA type was observed in the three myeloid cell lines. Except for the normal LCL, no B-lymphoid lineage related cell lines of various levels of differentiation displayed PA secretion, whereas PA activity was observed in the supernatant of six of nine malignant T-cell lines. The T-leukemic cell lines CCRF CEM, KE 37, HUT 78, and HUT 102 released U-PA-like activity. Peer released t-PA-like activity and CCRF-HSB2 supernatant showed both types of PA activity. These findings are discussed in view of the natural history of these diseases and the stage of differentiation of the cell lines.

Epstein-Barr virus-positive and -negative B-cell lines can be infected with human immunodeficiency virus types 1 and 2.

Human immunodeficiency virus type 1 (HIV-1) can infect CD4+ lymphocytes, monocytes-macrophages, and various other cell lines, including B-cell lines. To study the parameters of B-cell infections, we examined the susceptibility of 24 B-lymphoid cell lines to both HIV-1 and HIV-2 infections. These cell lines included a series of Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma cell lines and their EBV-converted counterparts. To infect these cells we used two HIV-1 isolates and one HIV-2 isolate. Infections were monitored with a cytoplasmic RNA dot-blot and a syncytium assay. HIV infection was also studied by a novel method based on electrophoresis of DNA liberated from cells that were lysed in situ in the well of an agarose gel. All human B-cell lines could be infected with HIV-1, regardless of the presence of EBV genomes; thus, EBV infection had no major effect on HIV susceptibility of B-cell lines. Integrated proviral HIV genomes could be detected by Southern blot analysis of DNA extracted from long-term, non-HIV-producing B-cell lines. This study suggests that B-lymphoid cells may serve as reservoirs for latent or persistent HIV infections in vivo, even in the absence of EBV infection.

Differential effects of benzamidine derivatives on the expression of c-myc and HLA-DRα genes in a human B-lymphoid tumor cell line

In this paper, we report the effects of aromatic tetra amidines (TAPP-H) on cell growth and gene experession of a B-lymphoid human tumor cell line, WI-L2. The results obtained give evidence (a) for inhibition of cell proliferation by TAPP-H; (b) for stronger antiproliferative activity of TAPP-halo derivatives; (c) for TAPP-mediated inhibition of accumulation of c-myc RNA sequences but not of HLA-DRα mRNA and DR antigens. These results suggest that this class of antiproliferative compounds exibit differential effects on cell-cycle specific and differentiation specific genes. In addition, also TAPP-related compounds containing 2 (DAPP) or 3 (TAPB) benzamidine residues retain inhibitory activity on the proliferation of WI-L2 cells. These latter compounds might be proposed as useful substrate in the synthesis of drug-conjugated monoclonal antibodies or growth factors.

Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes.

Four distinct human B-lymphoid cell lines possess the ability to circumvent the mechanism regulating intracellular transport of immunoglobulin protein. These cells do not produce light chains, yet they express mu heavy chains on the cell surface at comparable levels to B-cell lines that produce native forms of both proteins. The mu-chain mRNA produced in all four cell lines was found to contain an identical deletion of most of the heavy-chain variable (VH) region (75% of the 3' portion), with no apparent alteration in constant (C) region structure. The truncated mu (mu*)-chain mRNA in these cells was created through the use of a cryptic splice donor site found within the human VH gene(s) utilized by these B-cell lines. The truncated mu chains exhibited a decreased ability to associate with the intracellular transport regulatory protein, heavy-chain binding protein (BiP). This result indicates that VH region structure, in addition to C mu 1 region structure, influences the formation of the BiP recognition site on the heavy chain. Furthermore, it suggests that the mechanism allowing for cell-surface expression of the mu* chains in the absence of light-chain pairing is the inability of BiP to bind to the mu* chains and hence prevent their intracellular transport. The high frequency with which the mu-only surface immunoglobulin positive phenotype is present in our collection of human B-cell lines and the isolation of one of the cell lines from a healthy individual also suggest that B cells of this type may represent a significant subpopulation among the normal human B-cell repertoire.