Cyclin D3 at 6p21 is dysregulated by recurrent chromosomal translocations to immunoglobulin loci in multiple myeloma.

Reciprocal chromosomal translocations, which are mediated by errors in immunoglobulin heavy chain (IgH) switch recombination or somatic hypermutation as plasma cells are generated in germinal centers, are present in most multiple myeloma (MM) tumors. These translocations dysregulate an oncogene that is repositioned in proximity to a strong IgH enhancer. There is a promiscuous array of nonrandom chromosomal partners (and oncogenes), with the 3 most frequent partners (11q13 [cyclin D1]; 4p16 [FGFR3 and MMSET]; 16q23 [c-maf]) involved in nearly half of MM tumors. It is now shown that a novel t(6;14)(p21;q32) translocation is present in 1 of 30 MM cell lines and that this cell line uniquely overexpresses cyclin D3. The cloned breakpoint juxtaposes gamma 4 switch sequences with 6p21 sequences that are located about 65 kb centromeric to the cyclin D3 gene. By metaphase chromosome analysis, the t(6;14) (p21;q32) translocation was identified in 6 of 150 (4%) primary MM tumors. Overexpression of cyclin D3 messenger RNA (mRNA) was identified by microarray RNA expression analysis in 3 of 53 additional primary MM tumors, each of which was found to have a t(6;14) translocation breakpoint by interphase fluorescence in situ hybridization analysis. One tumor has a t(6;22)(p21;q11) translocation, so that cyclin D3 is bracketed by the IgL and IgH breakpoints. These results provide the first clear evidence for primary dysregulation of cyclin D3 during tumorigenesis. It is suggested that the initial oncogenic event for most MM tumors is a primary immunoglobulin translocation that dysregulates cyclin D1, cyclin D3, and other oncogenes to provide a proliferative stimulus to postgerminal center plasma cells.

Comparison of therapy detection times between implantable cardioverter defibrillators with standard dual- and single-chamber pacing.

Previous implantable cardioverter defibrillators (ICDs) required patients in need of dual-chamber (DDD) pacing for improved hemodynamic status to undergo implantation of separate devices to treat bradycardia and/or ventricular arrhythmias. An investigation was conducted to verify the performance of a new ICD that combines both therapies.Sixty-nine patients at 17 European and Canadian centers were implanted with VENTAK AV models 1810/1815, ICD's that includes DDD pacing and algorithms designed to differentiate between atrial and ventricular arrhythmias. 36 of the cohort were compared to 32 patients tested at six centers with an external test device (VENTAK MINI). In both cohorts detection times were calculated for ventricular fibrillation (VF) induced at implant. The mean detection times (DT) from the VENTAK AV device were compared to the DT from the VENTAK MINI device. Patient characteristics of the VENTAK AV and the VENTAK MINI control groups were similar. Mean VF detection time (+/-SD) with the VENTAK AV device was 2.21 +/- 0.54 seconds, as compared with 1.87 +/- 0.62 seconds with the VENTAK MINI (p < 0.01), indicating that the difference in means did not exceed one second. The VENTAK AV system function did not demonstrate interaction with the pacemaker function, as indicated by the clinical significance with the detection times of the study device. The difference in detection times between cohorts did not statistically exceed one second. Appropriate detection of the new ICD was not compromised by the addition of the dual-chamber pacing therapy.

Alternatively spliced pp52 mRNA in nonlymphoid stromal cells.

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.

Early rearrangements of genes encoding murine immunoglobulin kappa chains, unlike genes encoding heavy chains, use variable gene segments dispersed throughout the locus.

Immunoglobulin heavy-chain variable region (TH) gene segments located closest to the joining (JH) gene segments are preferentially rearranged during ontogeny, indicating that chromosomal position influences the frequency of rearrangement. In addition, certain VH gene segments are repeatedly rearranged, suggesting that the DNA sequence or structure surrounding these segments may increase the probability of rearrangement. To determine whether there is similar based rearrangement of kappa variable (V kappa) gene segments, 25 rearrangements were sequenced from murine fetal and neonatal B-cell hybridomas and from subclones of a pre-B cell line that rearranged V kappa genes during in vitro culture. Four gene segments were isolated twice and one gene segment was isolated three times, suggesting that the process that targets individual variable gene segments for repeated rearrangement operates on both the VH and V kappa loci. Based on a current map of the V kappa locus, the rearranged gene segments belong to nine families that are dispersed throughout the locus. Thus, in these cell types, V kappa rearrangements use germ-line gene segments located across the entire locus, whereas the corresponding VH rearrangements use gene segments proximal to the JH gene segments. Heterogeneity of V kappa rearrangements would add diversity to the biased pool of VH rearrangements, producing a broad repertoire of antibodies early in development.

Cytotoxicity, uptake, polyglutamate formation, and antileukemic effects of 8-deaza analogues of methotrexate and aminopterin in mice.

In contrast to methotrexate (MTX) and aminopterin (AMT), the 8-deaza analogues of these antifolates are not substrates for rabbit liver aldehyde oxidase. Since they are not converted to 7-hydroxy derivatives, they have been investigated with regard to their cytotoxicity for CCRF-CEM cells, transport into these cells, and conversion to polyglutamate forms. For this purpose 3H-labeled analogues were synthesized. The drug concentrations of the analogues required to inhibit cell growth by 50% are significantly lower than for the parent compounds particularly for a short exposure of cells to the drug. Vmax and Km for unidirectional influx do not differ greatly among the four drugs, but amounts of uptake over 1 h are markedly different and increase in the order MTX less than 8-deazaMTX less than AMT less than 8-deazaAMT. During 1 h of uptake a much greater proportion of the 8-deaza analogues is converted to polyglutamate forms than in the case of parent drugs. Only 52% of MTX is converted to polyglutamates, whereas in the case of the other three compounds the conversion is greater than or equal to 90%. However, MTX is relatively efficient in adding two glutamate residues, whereas the other drugs predominantly accumulate as forms with only one additional glutamate (+Glu1). During 1 h of efflux the drugs without additional glutamates decrease to low concentrations and there is also a major loss of +Glu1 form, but there is also an increase in longer chain forms, especially in the case of MTX. The net result is a still greater disparity in total intracellular levels of the four drugs after the period of efflux. MTX has much lower toxicity in mice in vivo than the other three compounds, 8-deazaAMT being the most toxic. At the maximum tolerated dose MTX produced a considerably greater increase in life span for mice bearing P388 than any of the other drugs, and a somewhat greater increase for mice bearing L1210. Thus the 8-deaza analogues do not offer a therapeutic advantage over MTX against leukemias in the mouse, primarily due to their much greater toxicity.

Hormonal regulation of preputial gland function in male Microtus montanus, the montane vole.

1. Preputial gland function in male Microtus montanus is androgen-dependent, both in terms of preputial weights and in the production of a series of lipids which are present in M. montanus and absent from Microtus pennsylvanicus. 2. Production of these species-typical lipids is decreased but not eliminated in castrates, as well as in adrenalectomized castrates treated with corticosterone. Therefore, in the total absence of androgens, a low level of these lipids is still produced. 3. 5 alpha-dihydrotestosterone and 17 beta-estradiol enanthate have limited effects on maintenance of preputial weight, suggesting that testosterone itself is the active steroid in the preputial gland of this species.

Effects of cytotoxicity of 2-chloro-2'-deoxyadenosine and 2-bromo-2'-deoxyadenosine on cell growth, clonogenicity, DNA synthesis, and cell cycle kinetics.

The cytotoxic effects of the adenosine deaminase resistant analogues 2-bromo-2'-deoxyadenosine (2-BrdAdo) and 2-chloro-2'-deoxyadenosine (2-CldAdo) have been compared with those of deoxyadenosine (dAdo). Like 2-CldAdo, 2-BrdAdo is highly effective in inhibiting the growth of many T-lymphoblastoid, B-lymphoblastoid, and myeloid cell lines in culture. Concentrations required to inhibit growth of CCRF-CEM human T-lymphoblastoid cells by 50% (IC50) are: 2-CldAdo, 0.045 microM; 2-BrdAdo, 0.068 microM; dAdo, 0.9 microM in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine. Like dAdo, 2-BrdAdo causes a much greater decrease in DNA synthesis than in RNA and protein synthesis. For each of the nucleosides the concentration required to cause 50% inhibition of DNA synthesis (as measured by thymidine incorporation) in an 18-h exposure is very similar to the IC50 for growth and to the concentration required to decrease viability (clonogenicity) over 18 h by 50% (EC50). A fraction of CCRF-CEM cells (approximately equal to 30%) is resistant to killing by exposure to 2-BrdAdo or 2-CldAdo for 4 h at concentrations 100 times the EC50, but 3% of cells are resistant to exposure for 4 h to a concentration of dAdo 3 times the EC50. Each of the three nucleosides causes accumulation of cells in S phase, the accumulation becoming more marked with longer periods of exposure and with higher concentrations of nucleoside. During exposures for 18-24 h at a concentration of nucleoside near the EC50 most cells accumulate in S, with most in early S, whereas exposure to concentrations greater than EC95 accumulates cells at the G1/S border. This suggests that loss of viability is associated with a blockade of some process specifically occurring at the initiation of S phase. At an optimum dosage schedule, 2-BrdAdo and 2-CldAdo have similar therapeutic effects against L1210 in vivo, both producing over 99% cell kill, but the optimum dosage of 2-CldAdo is lower.

Recruitment patterns in the rat hindlimb muscle during swimming.

Cycle periods and electromyographic (EMG) burst durations of selected extensors and flexors are shorter during swimming than during running at 27 m/min, while the relative ON-OFF timing of these interrelationships are similar. The mean EMG activities of the tibialis anterior and adductor longus were higher and the soleus and medial gastrocnemius were lower during swimming than during running. Both fast and slow extensors are activated during both forms of locomotion, thus demonstrating that there is not a selective recruitment of fast or selective inhibition of slow ankle extensors during swimming as measures of muscle blood flow have suggested.

A conserved sequence in the immunoglobulin J kappa-C kappa intron: possible enhancer element.

Several functionally important genetic elements (such as the TATA box, mRNA splice sequences, poly(A) addition signal) were first detected as short segments of unexplained sequence homology within non-coding regions of different genes. A short region of unknown sequence in the intron between the human J kappa and C kappa immunoglobulin coding regions was found to be sufficiently homologous to the corresponding segment of the mouse gene to form stable heteroduplexes. Although no specific function has yet been definitely ascribed to this region (which we call the kappa intron conserved region, or KICR), some functional significance has been inferred from the findings that (1) activation of B lymphocytes induces a DNase hypersensitivity site in this region and (2) deletions including this region reduce expression of kappa genes introduced into lymphoid cells. To delineate the KICR more precisely and to test the generality of the sequence conservation in a third species, we have sequenced this region of the human and mouse genes and have examined the corresponding region of a recently cloned rabbit kappa gene. We find a segment of about 130 base pairs (bp) that shows striking conservation in all three species, demonstrating homology significantly higher than within the C kappa coding region itself. Two short sequences from the J kappa-C kappa intron that were noted by other investigators to be homologous to proposed 'enhancer' sequences both lie within the conserved region.

Gene segments encoding transmembrane carboxyl termini of immunoglobulin gamma chains.

In cell lines producing IgM, secreted and membrane bound forms of immunoglobulin mu heavy chains are produced from two separate mu mRNAs that are identical except for alternative 3' coding sequences. We now show that cell lines producing IgG likewise contain two mRNA species for immunoglobulin gamma chains. The major, 1.7 kilobase (kb) species encodes secreted gamma chains. A less abundant species of 3-4 appears to encode membrane bound gamma chains, in that it contains an alternative 3' end encoded in separate exons 3' to the remainder of the gene. The first exon of this M gene segment has been identified in chromosomal gamma 1 and gamma 2b gene clones by its sequence homology with the corresponding exon in the mu gene. Like the mu M exon, it encodes a probable transmembrane polypeptide segment. The flanking DNA sequences show a patchwork pattern of homology between genes that suggests a checkered evolutionary history.

RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene.

Both C kappa regions in MPC 11 cells are rearranged into active transcripion units, one producing a normal kappa chain and the other an internally deleted kappa fragment lacking a V region. The gene coding for the kappa fragment mRNA is aberrantly rearranged and lacks a site for V leads to C kappa splicing. An alternative splicing event which deletes the V region from the nuclear RNA precursor generates the kappa fragment mRNA.