Prognostic significance of the S-phase fraction of light-chain-restricted cytoplasmic immunoglobulin (cIg) positive plasma cells in patients with newly diagnosed multiple myeloma enrolled on Eastern Cooperative Oncology Group treatment trial E9486.

The bone marrow plasma cell labeling index (PCLI) as measured by bromodeoxyuridine uptake is a well-established independent prognostic factor for patients with newly diagnosed multiple myeloma, but the test is not easily done in most laboratories. The purpose of this study was to determine if the proliferative activity (% S-phase) as determined by two-color flow cytometry for cytoplasmic immunoglobulin (cIg) light chain and DNA content also had prognostic significance. As part of Eastern Cooperative Oncology Group clinical trial E9486, 500 patients had successful performance of the bone marrow PCLI. Of 349 patients who had flow cIg and DNA content cytometry, 210 had adequate data to reliably calculate S-phase %. Patients with low % S-phase fraction (<2%) had a significant overall survival advantage over patients high % S-phase fraction (>/=2%), median survivals 4.1 vs. 2.9 years (P = 0.032). Measurement of the S-phase % by flow cytometry gives significant prognostic information in patients with newly diagnosed myeloma. However, in multivariate analysis, S-phase % did not add prognostic information when PCLI was in the model. S-phase % added prognostic information only when all cases with flow measurement of S-phase % were included, and when PCLI was excluded from the model. Discriminating a population of only cIg positive cells proved difficult in patients with a low percentage of bone marrow plasma cells. Methodology to measure S-phase % in patients with a low percent plasma cells is needed before this technique can be used for diagnosis and prognosis in myeloma.

Coordinate transcription and V(D)J recombination of the kappa immunoglobulin light-chain locus: NF-kappaB-dependent and -independent pathways of activation.

To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor beta is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, including activation of NF-kappaB. To address the functional role of NF-kappaB in LPS and IFN-gamma induction of these events, we blocked the nuclear translocation of NF-kappaB by overexpression of a dominant negative mutant of IkappaB-alpha (IkappaB deltaN). Overexpression of the IkappaB deltaN protein results in an inhibition of LPS but not IFN-gamma activation of germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination. Our results demonstrate that activation of NF-kappaB is necessary but not sufficient for LPS activation of transcription and recombination at kappa. These results also suggest that NF-kappaB is not required for IFN-gamma activation of transcription or recombination. These results are important in establishing that there are multiple independent pathways of activation of both transcription and recombination.

Organization of the transcription factor binding sites in the kappa Ig intron enhancer. Effects of position, orientation, and spacing.

The kappa Ig intron enhancer is comprised of multiple sequence motifs known to bind trans-acting factors that activate gene expression. A species comparison reveals a high level of conservation of the organization of the transcription factor binding sites within the enhancer. The importance of the conserved organization of the kappa intron enhancer was examined by using topologic mutations that disrupt the position, orientation, and spacing of individual binding sites within the enhancer. The effects of these changes were monitored by their effects on reporter gene activity at two distinct stages of B cell development. Previously, mutational analysis indicated the kappa B and kappa E2 sequence motifs to be the most crucial sites for intron enhancer function. We have demonstrated that intron enhancer activity is dependent on the position of the kappa B and kappa E2 sequence motifs within the enhancer. Intron enhancer function is, however, independent of kappa B and kappa E2 binding site orientation and is flexible in spacing requirements among binding sites.

Enhancer mediated suppression of epsilon heavy-chain gene expression in a murine IgE-producing hybridoma.

In vitro co-culture of IgE-secreting hybridoma cells (B53) with spleen cells harvested from mice with established B53 tumours results in a specific, T cell-dependent suppression of epsilon-chain expression in the B53 cells. The role of immunoglobulin enhancers in the suppression of IgE synthesis in B53 cells was examined by transfecting B53 cells with CAT expression vectors containing the immunoglobulin heavy- or kappa light-chain intron enhancers or a Rous sarcoma virus (RSV) LTR. When epsilon-chain expression of transfected cells was suppressed in vitro. CAT expression was also suppressed in cells transfected with vectors containing the immunoglobulin heavy-chain gene enhancer, but not in cells transfected with vectors containing the kappa enhancer or RSV LTR. Thus, the T cell-dependent suppression of IgE synthesis in B53 cells correlates with a specific inactivation of the immunoglobulin heavy chain enhancer, strongly suggesting that T cell-mediated suppression of Ig synthesis can normally occur through specific repression of Ig enhancer function. This represents a new regulatory pathway involved in the control of IgE synthesis and is the first indication that the enhancer mediated expression of Ig genes in B cells can be modulated through T cell-dependent processes.

In vivo and in vitro regulation of IgE production in murine hybridomas.

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.

Novel recombinations of the IG kappa-locus that result in allelic exclusion.

Allelic exclusion of Ig H and L chain gene loci serves to ensure that a B cell expresses a single specificity antibody. The analysis of Abelson murine leukemia virus transformed cells that rearrange the kappa-locus during growth in cell culture has provided the opportunity to characterize intermediate steps in Ig gene rearrangement. By sequential cloning of an Abelson murine leukemia virus transformed cell line we have observed a novel two-step pathway that results in a rearrangement of a V kappa gene segment into the J-C kappa intron. This type of rearrangement effectively excludes functional kappa expression from that allele. A truncated mRNA product resulting from the V kappa signal exon splicing to the C kappa exon is diagnostic of these unique rearrangements. In addition to demonstrating a novel mechanism for allelic exclusion, the two-step pathway described serves to explain how V-intron recombination products were generated in previously described cell lines.

Initiation and processing of two kappa immunoglobulin germ line transcripts in mouse B cells.

The splicing patterns and sequences of two processed kappa immunoglobulin germ line mRNAs are presented. A 1.1-kilobase (kb) mRNA appeared to be derived from splicing of the previously characterized 8.4-kb germ line transcript, while a 0.8-kb mRNA was the splice product of a second 4.7-kb germ line transcript that initiated 50 base pairs upstream of J kappa 1. The interaction of the two kappa germ line promoters with nuclear binding factors is also examined. The potential role of these germ line transcripts in establishing the rearrangement potential of the locus is discussed.

The frequency of multiple recombination events occurring at the human Ig kappa L chain locus.

Products of Ig kappa L chain gene rearrangement in a variety of human B cell samples were investigated by sequential Southern blot hybridization analysis. By application of four region-specific probes (C kappa, J kappa, U' kappa and kappa de) a complete spectrum of kappa rearrangements, including both predicted and novel products, were detected. Nearly 30% of the products detected reflect multiple recombination of the kappa locus. The kappa-deleting element was responsible for 70% of the multiple rearrangements that were detected. Interestingly, eight kappa-expressing samples exhibited rearrangement of the kappa-deleting element. The remaining multiple recombination products were characteristic of double V kappa-J kappa rearrangement. This frequency reveals that secondary V-J rearrangement may significantly contribute to the expression of kappa L chains in humans.

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed.

Identification of a germ line transcript from the unrearranged kappa gene in human B cells.

A novel kappa immunoglobulin-hybridizing mRNA in cell lines derived from human B cells arrested at several stages of development has been identified. Hybridization studies demonstrate that this 1.5-kilobase mRNA species is the spliced product of a precursor germ line transcript initiating upstream of the unrearranged JKappa locus.

Single copy gene detection requiring minimal cell numbers.

There has been an increasing application of molecular DNA probes to evaluate a variety of clinical conditions. Frequently, the amount of tissue or number of cells available limits analysis by conventional DNA extraction and Southern blot hybridization. Moreover, DNA amplification techniques cannot be used in all cases. We have applied a modification of the DNA extraction-Southern blot hybridization technique to clinical samples which provides essentially quantitative recovery and analysis of DNA from minimal numbers of cells. DNA was obtained from cells which were immobilized in agarose blocks for lysis, deproteinization and restriction enzyme digestion. The DNA was then run directly into agarose gels to size fractionate for Southern blot analysis. Cells can be suspended in agarose blocks for over one year and frozen cells can be thawed and suspended in agarose. A variety of restriction enzymes can be used. Single copy sequences can be detected from as few as 5 x 10(4) cells. We have employed this method to examine immunoglobulin gene rearrangements in PBL from leukemia patients as well as bone marrow from myeloma patients. In addition, we have used the technique to accurately assess bone marrow engraftment after transplant. These results demonstrate a diagnostic application of this technique in a variety of clinical samples where there may be limited availability of cells.

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells.

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.

Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

Thymus-dependent in vivo suppression of IgE synthesis in a murine IgE-secreting hybridoma.

In previous studies we demonstrated that BALB/c mice bearing ascitic tumors of the IgE-secreting hybridoma B53 (epsilon, kappa, anti-dinitrophenyl) developed large numbers of Lyt-1-2+ Fc epsilon R(+) T lymphocytes (T cells with membrane Fc receptors) in response to the elevated serum IgE concentration. The development of Fc epsilon R(+) T lymphocytes was followed by a progressive decrease in the levels of serum IgE in spite of continued proliferation of the hybridoma cells. This sequence of events suggested that the IgE-secreting hybridoma triggered a suppressive immunoregulatory circuit of the host that inhibited IgE expression by the hybridoma cells. The present studies were undertaken to investigate the basis for the subsequent decline in serum IgE levels in mice with B53 tumors and to identify host factors that might be involved in this process. We observed that ascitic B53 cells recovered at increasing time points from BALB/c mice exhibited a selective decline in steady state levels and rates of synthesis of epsilon-heavy chain protein and mRNA. The expression of kappa-light chain protein and mRNA appeared relatively unchanged. The decrease in epsilon-heavy chain gene expression did not occur when B53 tumors were passaged in nu+/nu+ mice or in BALB/c mice depleted of Lyt-2+ cells (suppressor/cytotoxic cell lineage), but did occur in nu+/nu+ mice reconstituted with neonatal BALB/c thymus and in BALB/c mice depleted of L3T4+ cells (helper/inducer cell lineage). That Fc epsilon R(+) T lymphocytes were directly involved in the inhibition of IgE expression was supported by the earlier and more pronounced inhibition of B53 IgE in mice infused with Fc epsilon R(+) T lymphocytes. We conclude from these findings that: 1) the decline in serum IgE levels that occurs toward the end of each generation of in vivo passage of the B53 hybridoma is due to decreased production of IgE by the hybridoma cells, 2) the decreased production of IgE is due to a selective loss of epsilon mRNA expression, 3) the decrease production of IgE by B53 cells is dependent on the presence of Lyt-2+ cells, and 4) Fc epsilon R(+) T lymphocytes participate in the mechanism by which IgE production is suppressed.

In vitro splicing of kappa immunoglobulin precursor mRNA.

The in vitro splicing of kappa immunoglobulin precursor mRNA was studied as an example of a naturally occurring mRNA possessing multiple 5' splice sites. Several kappa mRNAs were generated in vitro by using an SP6 transcription system and were spliced in nuclear extracts derived from HeLa cells. Products and intermediates resulting from in vitro splicing were identified and characterized. In contrast to the in vivo situation, in which apparently only the 5'-most splice donor site is used, all of the 5' splice sites were used in vitro with equal frequency. Neither the presence or absence of variable region coding sequences nor the deletion of intron sequences had an effect on in vitro splice site selection.

Double recombination of a single immunoglobulin kappa-chain allele: implications for the mechanism of rearrangement.

DNA fragments containing immunoglobulin kappa-chain sequences from two different plasmacytomas (PC 3609 and PC 7043) were found by blot-hybridization studies to be dissociated from germ-line sequences on both the 3' and 5' ends. These fragments were cloned, sequenced, and found to contain the structural features of a product of two recombination events. Each contained a variable (V kappa) gene segment recombined with a joining (J kappa) gene segment followed by the characteristic kappa light chain V-J reciprocal structure, a 5' J kappa flanking sequence joined to a 3' V kappa flanking sequence. These segments of DNA represent double recombination products (DRPs) of the same kappa-chain allele. The DRP from PC 3609 contains a normal V-J1 recombination, while the DRP from PC 7043 contains an aberrant V-J2 recombination, resulting in a frameshift. The reciprocal structure in the PC 3609 DRP is the result of a V-J2 recombination; the reciprocal structure in the DRP of PC 7043 is the result of a V-J3 recombination and appears to have been derived directly from the productive kappa-chain gene recombination in that plasmacytoma. These products demonstrate the capacity of a single kappa light chain immunoglobulin allele to undergo multiple V-J recombinations. Furthermore, the presence of a V-J recombination and its reciprocal product in the same cell is inconsistent with a segregating mechanism, such as sister chromatid exchange, but is consistent with an inversion mechanism.

DNA between variable and joining gene segments of immunoglobulin kappa light chain is frequently retained in cells that rearrange the kappa locus.

A systematic analysis of the fate of the DNA between kappa chain variable (V kappa) and joining (J kappa) genes in cells that have rearranged kappa loci was carried out. The DNA from a variety of kappa-producing plasmacytomas, lambda-producing hybridomas, and kappa-expressing lymphocytes was digested, fractionated by size, and analyzed with two probes containing sequences 5' of J kappa. In 13 of 28 plasmacytomas examined the rearrangement of V kappa and J kappa appears to be accompanied by loss of DNA upstream of J kappa. However, in the rest of the plasmacytomas one or more upstream sequences are retained in a new context. In 9 of 12 lambda-producing hybridomas (which frequently rearrange both kappa loci) one or more upstream segments were detected. These unique fragments were probably generated by a recombination event near or at the J kappa region. The extent to which the region between V and J is maintained in kappa-expression lymphocytes was also measured. Most (76%) of the region upstream of J kappa is retained in the population, even though 68% of the kappa loci are rearranged. In order to explain how these upstream elements occur in some, but not all, cell lines, and the significant occurrence in the lymphocyte population, we propose a model in which a step in V--J joining involves mitotic recombination by unequal sister chromatid exchange.

Transcription of the unrearranged mouse C kappa locus: sequence of the initiation region and comparison of activity with a rearranged V kappa-C kappa gene.

In cells of the B-lymphocyte lineage, 8.4 kb transcripts are constitutively produced from unrearranged kappa constant region (kappa 0) loci. To help elucidate the molecular basis of this phenomenon, we have determined the nucleotide sequence surrounding the site of transcriptional initiation. The kappa 0 transcripts are initiated within a unique Eco RI fragment located about 8 kb upstream from the C kappa gene. The start site is about 36 nucleotides downstream from a Hogness consensus sequence (TGTAAAT) and nearly 200 nucleotides upstream from a sequence that is similar to those encoding the signal peptides of kappa light chains. These features, which are usually found in the 5' flanking regions of kappa variable region genes, suggest that the kappa 0 initiation sequence may be an evolutionary relic of some common ancestral 5' element. In contrast, there is no discernible V kappa-encoding element in 780 nucleotides of sequence downstream from the initiation site. From pulse-chase-labeling experiments with a pre-B-cell hybridoma line and direct measurements of transcriptional activity in isolated nuclei, we have estimated that the rate of transcription of the kappa 0 locus is significantly lower than that of a rearranged V kappa-C kappa gene. This result, together with the fact that unrearranged V kappa genes are transcriptionally silent, suggests that structural features of both the V kappa and C kappa loci contribute to the overall transcriptional efficiency of a rearranged V kappa-C kappa gene. The 8.4 kb transcripts are not processed into any stable RNA products, despite the fact that they contain some apparently normal splice junctions; rather, they are degraded within the nucleus at about half the rate with which a kappa mRNA precursor is processed. Conceivably, the transcriptional activity of the kappa 0 locus might be a prerequisite for its recombinatorial activity.