Genetic evidence that Sil is required for the Sonic Hedgehog response pathway.

The Sil gene encodes a cytosolic protein required for mouse embryonic midline and left/right axial development. Based on the phenotype of Sil mutant embryos, we hypothesized that Sil may be required for the activity of Sonic Hedgehog (Shh), a secreted signaling molecule also critically important for the development of the embryonic axes and found mutated in multiple types of cancer. Here we tested the genetic interaction between Sil and the Shh pathway by generating and analyzing embryos carrying mutations in both Sil and Patched (Ptch), a Shh receptor that normally inhibits the signaling pathway in the absence of ligand and when mutated leads to constitutive activation of the pathway. We find that Sil(-/-) Ptch(-/-) embryos do not activate the Shh pathway and instead have a phenotype indistinguishable from Sil(-/-) embryos, in which there is a loss of activity of Shh. These results provide genetic evidence that Sil is an essential component of the Shh response, acting downstream to Ptch.

The SIL gene is required for mouse embryonic axial development and left-right specification.

The establishment of the main body axis and the determination of left-right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left-right anomalies. This association is also seen in a number of mutations in mouse and zebrafish, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left-right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2.

The absolute number of trans-rearrangements between the TCRG and TCRB loci is predictive of lymphoma risk: a severe combined immune deficiency (SCID) murine model.

Pilot studies in human populations have demonstrated a correlation between the level of antigen receptor trans-rearrangements and risk (at the population level) of lymphoid malignancy. Irradiation of newborn severe combined immune deficiency mice results in an increased risk of subsequent development of thymic lymphoma (100% of mice so irradiated are dead of thymic lymphoma by 20 weeks of age). We, therefore, assayed the occurrence of trans-rearrangements in this well-controlled mouse mutant system and found a 50-100-fold increase in the absolute number of TCRGV-TCRBJ trans-rearrangements compared to unirradiated littermates (and a comparable fold increase over age-matched BALB/c mice) at 2 weeks following irradiation. We also found a marked disproportion in generating trans-rearrangements versus intralocus rearrangements in the severe combined immune deficiency system compared to BALB/c, independent of irradiation. The trans-rearrangements noted were polyclonal in nature. These data, again, suggest that the absolute level of antigen receptor trans-rearrangements may serve as a biomarker of lymphoma risk.

Expression of the SIL gene is correlated with growth induction and cellular proliferation.

The SIL gene was discovered at the site of a cancer-associated interstitial deletion in which its promoter assumed the regulation of a second gene, SCL. The human SIL gene encodes a 1287-amino acid cytosolic protein that has been found to be highly conserved in the mouse. SIL is expressed in proliferating cells and is down-regulated when cellular proliferation ceases because of serum starvation, contact inhibition, or induction of terminal differentiation. SIL is induced within 1 h of stimulation by 20% serum in growth-arrested 3T3 cells. This induction is independent of protein synthesis because "superinduction" is observed in the presence of the protein synthesis inhibitor cyclohexamide. Thus, SIL is an immediate-early gene. Upon release from serum starvation of 3T3 fibroblasts, SIL mRNA and protein levels display a biphasic pattern during the first cell cycle. In contrast, in exponentially growing EL4 lymphoblasts, SIL mRNA is stable throughout the cell cycle, whereas SIL protein accumulates into G2 phase and then falls precipitously at the completion of the cell cycle. This pattern of cell cycle expression suggests that SIL may play an important role in cellular growth and proliferation.

Molecular characterization of NSCL, a gene encoding a helix-loop-helix protein expressed in the developing nervous system.

We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development.

Disruption of the human SCL locus by "illegitimate" V-(D)-J recombinase activity.

A fusion complementary DNA in the T cell line HSB-2 elucidates a provocative mechanism for the disruption of the putative hematopoietic transcription factor SCL. The fusion cDNA results from an interstitial deletion between a previously unknown locus, SIL (SCL interrupting locus), and the 5' untranslated region of SCL. Similar to 1;14 translocations, this deletion disrupts the SCL 5' regulatory region. This event is probably mediated by V-(D)-J recombinase activity, although neither locus is an immunoglobulin or a T cell receptor. Two other T cell lines, CEM and RPMI 8402, have essentially identical deletions. Thus, in lymphocytes, growth-affecting genes other than immune receptors risk rearrangements.

The SCL gene is formed from a transcriptionally complex locus.

We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically its 5' region, may be a target for regulatory interactions during early hematopoietic development.

Characterization of the breakpoint of a t(14;14)(q11.2;q32) from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia.

The leukemic cells and derivative cell line from a 74-year-old male with T-cell acute lymphoblastic leukemia showed chromosomal abnormalities including a t(14;14)(q11.2;q32). This translocation is characteristic of a variety of T-cell malignancies, particularly T-cell prolymphocytic leukemia and the clonal proliferations of peripheral T cells in patients with ataxia-telangiectasia. Using DNA probes that spanned the T-cell receptor alpha chain (TCRA) joining (J) locus, the DNA rearrangement caused by the translocation was identified, cloned, and sequenced. The breakpoint shows site-specific juxtaposition of a TCRA joining segment and DNA from a region of 14q32 centromeric to the immunoglobulin heavy chain locus. Comparison of restriction map and nucleotide sequence from this translocation with other related chromosomal breakpoints suggests a dispersion of breakpoints throughout the 14q32 region.

Juxtaposition of the T-cell receptor alpha-chain locus (14q11) and a region (14q32) of potential importance in leukemogenesis by a 14;14 translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia.

We describe a t(14;14)(q11;q32) translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia (AT). By using a battery of joining (J)-segment probes from the T-cell receptor (TCR) alpha-chain locus TCRA, three distinct J alpha rearrangements were observed. One rearrangement reflected a normal TCRA variable (V) region V alpha-to-J alpha recombination. The second rearrangement was caused by the translocation even itself, which joined a DNA segment from 14q32 centromeric to the immunoglobulin heavy chain locus (IGH) and a J alpha gene located approximately 75 kilobases (kb) 5' of the TCRA constant region gene (C alpha). A third rearrangement involved a 17-kb internal deletion 3' to the translocation, a rearrangement within the J alpha locus that has been observed once before in a patient with AT. Analysis of these three rearrangements underscores the increase in aberrant locus-specific recombination in lymphocytes from patients with AT. Furthermore, these studies support the view that a growth-effecting gene is present in the 14q32 region that participates in the leukemogenic process.

Complex translocation disrupts c-myc regulation in a human plasma cell myeloma.

A complex translocation has interrupted the third exon of the c-myc gene in human plasma cell myeloma tumor cells and a derivative cell line (NCI-H929). As a result of this rearrangement, a chimeric mRNA is expressed which commences 5' of the c-myc coding region and includes sequences introduced by the translocation event. All of the detectable c-myc-containing mRNA in the tumor and cell line was derived from this rearranged c-myc allele. This chimeric c-myc mRNA, in which most of the germ line c-myc 3' untranslated region has been replaced, was greater than sevenfold more stable than c-myc transcripts with intact 3' ends. This suggests that the 3' untranslated region may play an important role in c-myc mRNA stability.

Human gastrin-releasing peptide gene maps to chromosome band 18q21.

A complementary DNA clone encoding human pre-pro gastrin-releasing peptide, a 27-amino acid neuropeptide and putative growth factor, was used to determine the chromosomal location of this gene. Southern blot hybridization to genomic DNA isolated from a panel of human-rodent somatic cell hybrids unambiguously maps this gene to human chromosome 18. In situ chromosomal hybridization confirms the hybrid data and further localized the gene to chromosome band 18q21. Karyotypic abnormalities in tumors and inherited disease states which involve chromosome band 18q21 may now be studied for correlated changes in the structure and expression of the human GRP gene.

The human galactosyltransferase gene is on chromosome 9 at band p13.

The structural gene for galactosyltransferase (glycoprotein 4-B-galactosyltransferase, EC 2.4.1.38) was localized to human chromosome 9 band p13 by chromosome in situ hybridization using a cloned bovine galactosyltransferase cDNA probe. This chromosomal location is at the same position to which galactose-1-phosphate uridyltransferase, an enzyme which provides the nucleotide sugar substrate (UDP-galactose) for galactosyltransferase, has been mapped.

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation.

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.

L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer.

Altered structure and regulation of the c-myc proto-oncogene have been associated with a variety of human tumours and derivative cell lines, including Burkitt's lymphoma, promyelocytic leukaemia and small cell lung cancer (SCLC). The N-myc gene, first detected by its homology to the second exon of the c-myc gene, is amplified and/or expressed in tumours or cell lines derived from neuroblastoma, retinoblastoma and SCLC. Here we describe a third myc-related gene (L-myc) cloned from SCLC DNA with homology to a small region of both the c-myc and N-myc genes. Human genomic DNA shows an EcoRI restriction fragment length polymorphism (RFLP) of L-myc defined by two alleles (10.0- and 6.6-kilobase (kb) EcoRI fragments), neither associated disproportionately with SCLC. Mouse and hamster DNAs exhibit a 12-kb EcoRI L-myc homologue, which indicates conservation of the gene in mammals. Gene mapping studies assign L-myc to human chromosome region 1p32, a location distinct from that of either c-myc or N-myc but associated with cytogenetic abnormalities in certain human tumours. This L-myc sequence is amplified 10-20-fold in four SCLC cell line DNAs and in one SCLC tumour specimen taken directly from a patient. Either the 10.0- or 6.6-kb allele can be amplified and in heterozygotes only one of the two alleles was amplified in any SCLC genome. SCLC cell lines with amplified L-myc sequences express L-myc-derived transcripts not seen in SCLC with amplified c-myc or N-myc genes. In addition, some SCLCs without amplification also express L-myc-related transcripts. Together, these findings suggest an enlarging role for myc-related genes in human lung cancer and provide evidence for the concept of a myc family of proto-oncogenes.

T-cell receptor gene rearrangements as clinical markers of human T-cell lymphomas.

The ability to detect immunoglobulin-gene rearrangements has proved useful in confirming diagnoses of suspected B-cell lymphomas and in establishing their monoclonality. By analogy, we employed a cloned DNA probe for the beta chain of the T-cell receptor gene to determine whether gene rearrangements were present in human T-cell neoplasms representing various stages of T-cell development. Gene rearrangements were present in all cases of T-cell disorders except a single case of T gamma lymphocytosis, a disorder that has not been proved to be a clonal T-cell neoplasm. A germline gene configuration was present in all patients with non-T-cell neoplasms and in normal tissues from patients with T-cell lymphoma. The probe promises to be useful for confirming the pathological an immunologic diagnosis in difficult cases of T-cell disorders and for assessing the extent of disease.

T cell receptor alpha chain genes are located on chromosome 14 at 14q11-14q12 in humans.

A cDNA clone encoding the alpha chain of the human T cell receptor was used in connection with somatic cell human-rodent hybrids to determine that the genes coding for the alpha chain are located on chromosome 14 in humans. In situ hybridization confirms this result and further localizes these genes to 14q11-14q12 on this chromosome. Since this region of chromosome has been shown to be nonrandomly involved in a number of T cell neoplasias, this assignment raises a number of interesting questions as to the possible involvement of the T cell receptor alpha chain genes in tumorigenesis.

Bombesin-like peptides and receptors in human tumor cell lines.

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.