Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line.

External ear hole closure in LG/J mice represents a model of regenerative response. It is accompanied by the formation of a blastema-like structure and the re-growth of multiple tissues, including cartilage. The ability to regenerate tissue is heritable. An F34 advanced intercross line of mice (Wustl:LG,SM-G34) was generated to identify genomic loci involved in ear hole closure over a 30-day healing period. We mapped 19 quantitative trait loci (QTL) for ear hole closure. Individual gene effects are relatively small (0.08 mm), and most loci have co-dominant effects with phenotypically intermediate heterozygotes. QTL support regions were limited to a median size of 2 Mb containing a median of 19 genes. Positional candidate genes were evaluated using differential transcript expression between LG/J and SM/J healing tissue, function analysis and bioinformatic analysis of single-nucleotide polymorphisms in and around positional candidate genes of interest. Analysis of the set of 34 positional candidate genes and those displaying expression differences revealed over-representation of genes involved in cell cycle regulation/DNA damage, cell migration and adhesion, developmentally related genes and metabolism. This indicates that the healing phenotype in LG/J mice involves multiple physiological mechanisms.

A common mechanism of chromosomal translocation in T- and B-cell neoplasia.

The chromosomal breakpoint involved in the t(8;14)(q24;q11) chromosome translocation in the SKW-3 cell line, which directly involves the 3' flanking region of the c-myc gene, was cloned and sequenced. The breakpoint on chromosome 8 mapped to a position 3 kb 3' of c-myc while the chromosome 14 breakpoint occurred 36 kb 5' of the gene for the constant region of the alpha chain of the T-cell receptor (TCR). The translocation resulted in a precise rearrangement of sequences on chromosome 8 and what appears to be a functional J alpha segment on chromosome 14. Signal sequences for V-J joining occurred at the breakpoint positions on both chromosomes 14 and 8, suggesting that the translocation occurs during TCR gene rearrangement and that it is catalyzed by the enzymatic systems involved in V-J joining reactions. The involvement of c-myc in the translocation and the association of joining signals at the breakpoints provides a parallel to the situation observed in the translocations involving c-myc and the immunoglobulin loci in B-cell neoplasms and suggests that common mechanisms of translocation and oncogene deregulation are involved in B- and T-cell malignancies.

Cloning and sequencing of a c-myc oncogene in a Burkitt's lymphoma cell line that is translocated to a germ line alpha switch region.

We have cloned and sequenced the translocated c-myc gene from the Burkitt's lymphoma CA46 cell line that carries a reciprocal translocation between chromosomes 8 and 14. The breakpoint lies within the first intron of c-myc, so that the first noncoding exon of the gene remains on the 8q- chromosome. The second and third coding exons are translocated to the 14q+ chromosome into the switch region of C-alpha 1. The orientation of the c-myc gene with relationship to alpha 1 is 5' to 5', with directions of transcription in opposite orientation. DNA sequencing studies predict five changes in the amino acid sequence of the myc protein, two of which occur in a region within the second exon which is highly conserved in evolution. Southern blotting data indicate that the first exon of c-myc is rearranged 3' to 3' with the pseudo-epsilon gene. Because CA46 cells contain two rearranged mu genes, the translocation must have occurred after immunoglobulin rearrangement. The position of the breakpoint in CA46 occurs within a 20-base-pair region of the first intron of c-myc to which breakpoints have been mapped for two additional B-cell lymphomas with the t(8;14) translocation, ST486 and the Manca cell line. The region of the heavy chain locus to which c-myc has translocated is different in each case. Comparisons have been made of the levels of transcripts of the translocated c-myc gene in ST486 and CA46, where the gene is not associated with the heavy chain enhancer, with its expression in the Manca cell, in which it is. The c-myc gene is transcribed at similar levels in all three cases.

Translocations and rearrangements in T-cell acute leukemias with the t(11;14) (p13;q11) chromosomal translocations.

Chromosomal translocations involving the T-cell receptor alpha and delta genes at q11 on chromosome 14 are the most common cytogenetic abnormalities in patients with T-cell tumors. We have demonstrated that the t(11;14)(p13;q11) translocation in two T-ALL patients involves the J delta region suggesting that the translocation proceeds or coincides with delta gene rearrangement. Additional rearrangements on both normal and translocated chromosomes 14 are described including rearrangement of both Tcr-alpha and Tcr-delta genes, and deletions within the J alpha region. The polyclonality of rearrangements on the normal chromosome 14 in one of the patient samples demonstrates that T-cell receptor rearrangement continues after the translocation event. The identification of clonally expanded rearrangements involving both the Tcr-delta and the Tcr-alpha genes in a single patient suggests a cascade model for delta----alpha expression may be a viable pathway for T-cell maturation.

Effects of pro-inflammatory cytokines on cannabinoid CB1 and CB2 receptors in immune cells.

AIMS: To investigate the regulation of cannabinoid receptors CB1 and CB2 on immune cells by pro-inflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS). CB1 and CB2 signalling may be anti-inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB1 and CB2 expression and function are unknown. METHODS: Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB1 and CB2 mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression of CB1 and CB2 protein was determined by flow cytometry. CB1 and CB2 signalling in PBMC was determined by Western blotting for Erk1/2. RESULTS: Pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α (the latter likely NF-κB dependently) can upregulate CB1 and CB2 on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate upregulation of CB1 and CB2 and increased IL-1ß, IL-6 and TNF-α mRNA in blood of patients with MS compared with controls. CONCLUSION: The levels of CB1 and CB2 can be upregulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.

Dysregulation of lymphocyte interleukin-12 receptor expression in Sézary syndrome.

Initial phase I and II clinical trials with recombinant human interleukin-12 have demonstrated the therapeutic efficacy of this cytokine in early stage cutaneous T cell lymphoma as compared with more advanced stages such as the leukemic Sézary syndrome. In an effort to optimize the use of recombinant human interleukin-12, using flow cytometry we studied the regulation of the interleukin-12 receptor beta1 (high affinity chain) and beta2 (chain necessary for interleukin-12 signal transduction) on normal volunteer CD4+ and CD8+ T cells and CD4+ and CD8+ cells from eight patients with different degrees of leukemic involvement with Sézary syndrome. The beta1 chain was not readily detectable on resting normal and T cells from Sézary patients, but expression was induced following T cell activation with phytohemagglutinin. Similarly, the beta2 chain was not detectable on resting normal volunteer T cells, but could be induced following phytohemagglutinin stimulation. Moreover, the beta2 chain on normal volunteer T cells was markedly upregulated following short-term culture with interferon-gamma or recombinant human interleukin-12. CD8+ T cells routinely exhibited a greater expression of beta2 than did CD4+ T cells. In marked contrast, both CD4+ and CD8+ T cells from patients with Sézary syndrome and a high tumor cell burden (> 50% circulating atypical Sézary T cells) failed to express the beta2 chain under any culture conditions. Although, culture with anti-interleukin-10 also markedly increased beta2 expression on normal volunteer T cells, this failed to induce expression on either CD4+ or CD8+ T cells from Sézary patients and a high tumor burden. Investigation of patients with Sézary syndrome and a low tumor cell burden (< 15% circulating Sézary T cells) revealed a pattern of beta2 expression that was intermediate between advanced Sézary syndrome and normal volunteers. Both CD4+ and CD8+ peripheral blood T cells from these earlier stage patients were induced to express the beta2 chain, although at a lower frequency of positivity than T cells from normals, following culture with phytohemagglutinin, interferon-gamma, recombinant human interleukin-12, or anti-interleukin-10. These results indicate that short-term culture with interferon-gamma and recombinant human interleukin-12 potently upregulates beta2 chain expression on T cells from normal volunteers, whereas a similar, but less marked effect occurs on T cells from Sézary syndrome patients and a low circulating tumor cell burden. In contrast, the beta2 chain appears to be suppressed on both CD4+ and CD8+ T cells from Sézary patients with a heavy circulating tumor cell burden and it is not induced by interferon-gamma or recombinant human interleukin-12. Therefore, recombinant human interleukin-12 is likely to be most effective for early stage cutaneous T cell lymphoma due to a greater display of beta2 receptors on responding CD8+ anti-tumor cytotoxic T cells.

The role for interleukin-12 therapy of cutaneous T cell lymphoma.

Recent phase I and phase II trials using recombinant human interleukin-12 (rhIL-12) for cutaneous T cell lymphoma (CTCL) have been completed. Observations on 32 evaluable patients revealed an overall response rate approaching 50 percent. Biopsy of regressing lesions revealed an increase in numbers of CD8+ and/or TIA-1+ T cells. These results suggest that rhIL-12 may induce lesion regression by augmenting antitumor cytotoxic T cell responses. Future trials will be focused on strategies for further immune enhancement by the concomitant use of additional immune augmenting cytokines with rhIL-12.

The Rac1 splice form Rac1b promotes K-ras-induced lung tumorigenesis.

Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. Although expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.

Oncogene probes in the detection of human cancer.

Specific chromosomal translocations are involved in more than 80% of human B cell neoplasms. In all these cases, the neoplastic phenotype is apparently the consequence of reciprocal chromosomal translocations involving the loci for human immunoglobulin chains and either well-described cellular protooncogenes or putative protooncogenes. The juxtaposition of the protooncogenes to the immunoglobulin loci results in their transcriptional deregulation, because of their proximity to genetic elements within the human immunoglobulin loci capable of activating gene transcription in cis over considerable chromosomal distances. Sequence analysis of the translocation breakpoints has provided important insights concerning the molecular mechanisms involved in chromosome translocation in B cells. It appears that the reciprocal translocations contributing to B cell neoplasia are catalyzed by the same enzymes that are involved in physiological immunoglobulin gene rearrangements. The analysis of human B cell leukemias and lymphomas has also provided considerable information concerning the possible scenarios for B cell neoplastic transformation. It is clear that the Epstein-Barr virus does not play a direct role in neoplastic transformation, but it may contribute by increasing the number of B cells at risk of developing chromosome translocations during immunoglobulin gene rearrangements. Cytogenetic and molecular genetic analysis of T cell malignancies is beginning to provide a very similar scenario for neoplastic transformation. The locus for the alpha-chain of the T cell receptor is directly involved, and it apparently juxtaposes to protooncogenes or to putative protooncogenes leading to their transcriptional deregulation. It seems quite likely that the enzyme system involved in rearrangements of the genes for the T cell receptor plays a crucial role in the causation of these chromosomal translocations. Thus, the genetic basis of many human B and T cells may be quite similar. For the future, the challenge resides in trying to characterize specifically the role of both old and new protooncogenes in B and T cell proliferation, normal and neoplastic.

Localization of 14 functional J regions in the human T-cell receptor alpha-chain locus.

Nearly 60 unique J alpha regions have been identified in human T-cell receptor (TCR) alpha chains to date, yet fewer than one-third of these have been localized within the alpha-chain locus. We report a rapid method for the mapping of productively rearranged J alpha regions using oligonucleotide probes and overlapping phage clones spanning 80 kb of the TCR-alpha locus. Fourteen J alpha regions were mapped using this technique; the positions of five of these have been verified by genomic cloning.

Cloning and sequencing of a rearranged V lambda gene from a Burkitt's lymphoma cell line expressing kappa light chains.

We have cloned and sequenced a rearranged V lambda gene from a Burkitt's lymphoma cell line PA682(PB). This cell line has two rearranged kappa loci and has been shown to be expressing kappa light chains. This V lambda gene has been identified as a member of the V lambda subgroup III gene family based on the homology of the predicted amino acid sequence of PAV lambda with the reported sequences of the V lambda protein DEL of subgroup III. Nine cross-hybridizing bands have been detected on Southern blots and the chromosomal orientation of the V lambda subgroup III gene family has been determined in relation to the V lambda subgroup I gene family. Although the PAV lambda rearrangement has occurred via a legitimate V-J joining and a normal size transcript is detected on Northern blots, the nucleotide sequence reveals a high level of mutations resulting in multiple termination signals within the V gene coding sequence and only a truncated V lambda protein can be translated. This confirms previous observations that although multiple light chain genes may be transcribed, only one functional light chain protein can be synthesized.

Localization of the gene for the erythroid anion exchange protein, band 3 (EMPB3), to human chromosome 17.

We have isolated genomic DNA clones which code for the human erythroid membrane protein band 3 (EMPB3). The identification of the gene has been confirmed by comparison of the amino acid sequence derived from the nucleotide sequence for two restriction fragments from the 5' end of the gene. Two exons have been identified. One exon encodes 20 amino acids which are identical to residues 36 to 56 of the band 3 protein, and the other encodes 44 amino acids homologous to residues 118 to 162. Southern analysis of genomic DNA derived from a panel of rodent-human somatic cell hybrids, which retain different complements of human chromosomes, with band 3 probes has allowed us to localize EMPB3 to human chromosome 17. The gene has been further localized between 17q21 and qter by analysis of DNA from somatic cell hybrids which carry derivative chromosomes from translocations involving chromosome 17 and either chromosome 15 or 21.

Analysis of the 3' flanking region of the human c-myc gene in lymphomas with the t(8;22) and t(2;8) chromosomal translocations.

We have cloned and mapped the sequences extending 38 kb 3' of the c-myc gene. This region is found to be highly repetitive in nature and hybridizes extensively with a BLUR 8 Alu probe. Unique sequence probes derived from this region were used to map the chromosomal breakpoints of a number of lymphoma cell lines with t(2;8) or t(8;22) translocations. In five of the cell lines (PA682, LY67, LY47, LY66 and LY91), the immunoglobulin light chain locus translocates into a region which is greater than 47 kb downstream of c-myc. For one of the cell lines, JI, the location of the breakpoint on the 8q+ chromosome was found to be 25-32 kb 3' of c-myc. The breakpoint for the BL2 cell line had been previously mapped at 10 kb 3' of the c-myc oncogene. Analyses of steady-state levels of c-myc mRNA in cell lines with chromosomal breakpoints ranging from 10 kb to greater than 47 kb 3' of c-myc range from 0.5 to 10X the levels in lymphoblast controls. The different levels of c-myc transcripts is not a direct function of the distance between the c-myc gene and the translocated immunoglobulin light chain locus.

Quantitation of transient gene expression after electroporation.

The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.

MYC oncogene involved in a t(8;22) chromosome translocation is not altered in its putative regulatory regions.

We have cloned the translocation-associated MYC gene from the Burkitt lymphoma cell line (BL2) with a t(8;22) chromosomal translocation and have determined the nucleotide sequence of the first exon and of the 3' and 5' flanking regions, where sequences with putative regulatory functions have been identified. The nucleotide sequence of the 5' flanking region, which contains regions of DNase hypersensitivity and binding sites for putative regulatory proteins, is the same as that of the normal MYC. Accordingly, mutations in these regulatory regions are not required for the transcriptional deregulation of MYC in the BL2 cell line. The nucleotide sequence of the first exon is similar to that of the normal MYC [Gazin, C., Dupont de Direchin, S., Hampe, A., Masson, J. M., Martin, P., Stehelin, D. & Galibert, F. (1984) EMBO J. 3, 383-387] and has the coding capacity for a 188-residue polypeptide. However, six nucleotide changes that occur in the middle of this reading frame could result in amino acid substitutions. We also have cloned and sequenced the t(8;22) chromosomal breakpoint that is located 10 kilobases 3' of the MYC exon 3 and near the C lambda 3 gene on chromosome 22. Sequences with homology to immunoglobulin joining signals occur close to the breakpoint both on chromosome 8 and 22, providing further evidence that the immunoglobulin joining enzymes may be involved in the recombinations associated with a variety of chromosomal translocations in B and T cells.

Isolation and partial characterization of a 48-kDa protein which is induced in normal lymphocytes upon mitogenic stimulation.

A 48-kDa protein (p48) crossreactive with an antiserum directed against the 12 C-terminal amino acids of the human cellular myc gene-encoded protein was isolated from a Burkitt lymphoma cell line. The p48 protein is a basic protein and has a cytoplasmic localization. An antiserum prepared against purified p48 reacts specifically with a 48-kDa protein present in a variety of mouse and human cells. This polypeptide is detected at very low levels in normal, resting, peripheral blood lymphocytes, but is induced several-fold by stimulation with either concanavalin A or pokeweed mitogen. The association of p48 induction with a proliferative response and the crossreactivity with the cellular myc protein are discussed.

Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed.