atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia.

Eotaxin is an eosinophil-specific chemoattractant that has been recently identified in rodent models of asthma and host response against tumors. To determine whether a similar molecule might play a role in human inflammatory diseases characterized by eosinophilia, we isolated the human eotaxin gene. We demonstrate that human eotaxin is an early response gene of cytokine-stimulated epithelial and endothelial cells, and is induced in peripheral blood eosinophils by interleukin-3. Eotaxin is directly chemotactic for eosinophils, but not mononuclear cells or neutrophils. Eotaxin messenger RNA accumulates markedly in the lesions of patients with inflammatory bowel disease (ulcerative colitis and Crohn's disease), but not in the lesions of patients with diverticulitis. These results now provide a mechanism involving eotaxin to explain the eosinophil infiltration seen in a variety of human disease; as such, an eotaxin antagonist may be a novel therapy for certain human diseases characterized by tissue eosinophilia.

Transgenic expression of interleukin 7 restores T cell populations in nude mice.

The thymic lesion of the nude mouse causes a profound block in T cell development. The failure of most T cells to mature in nude mice is likely to reflect a requirement for signals elaborated in the normal thymus. Interleukin 7 (IL-7), a lymphokine that is normally expressed in the thymus and has been implicated in T cell maturation, might be central to this process. To test this possibility, we introduced a transgene directing lymphoid expression of IL-7 into nude mice and found that it substantially alleviates the block in T cell maturation caused by the thymic defect. IL-7 transgenic nude mice have increased numbers of peripheral cells expressing the T cell marker Thy-1, the T cell antigen receptor complex, and the co-receptors CD4 and CD8. The IL-7 transgene also restores T cell-specific proliferation and activation responses to the peripheral cells of transgene-rescued nude mice. Such findings point toward a fundamental role for IL-7 in the thymic maturation of T cells.

IP-10, a -C-X-C- chemokine, elicits a potent thymus-dependent antitumor response in vivo.

IP-10 is a member of the -C-X-C-chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). To date no function has been described for IP-10. We have genetically engineered tumor cells to secrete high levels of murine IP-10 and demonstrate that while IP-10 has no effect on the growth of these tumor cells in culture, it elicits a powerful host-mediated antitumor effect in vivo. The IP-10 antitumor response is T lymphocyte dependent, non-cell autonomous, and appears to be mediated by the recruitment of an inflammatory infiltrate composed of lymphocytes, neutrophils, and monocytes. These results document an important biologic property of IP-10 and raise the possibility that some of the T cell-directed effects of IFN-gamma and LPS may be mediated by this chemokine.

Expression of the mouse pre-T cell receptor alpha gene is controlled by an upstream region containing a transcriptional enhancer.

The pre-T cell receptor alpha (pTalpha) protein is a critical component of the pre-T cell receptor complex in early thymocytes. The expression of the pTalpha gene is one of the earliest markers of the T cell lineage and occurs exclusively in pre-T cells. To investigate the molecular basis of thymocyte-specific gene expression, we searched for the genomic elements regulating transcription of the mouse pTalpha gene. We now report that expression of the pTalpha gene is primarily controlled by an upstream genomic region, which can drive thymocyte-specific expression of a marker gene in transgenic mice. Within this region, we have identified two specific DNase-hypersensitive sites corresponding to a proximal promoter and an upstream transcriptional enhancer. The pTalpha enhancer appears to function preferentially in pre-T cell lines and binds multiple nuclear factors, including YY1. The enhancer also contains two G-rich stretches homologous to a critical region of the thymocyte-specific lck proximal promoter. Here we show that these sites bind a common nuclear factor and identify it as the zinc finger protein ZBP-89. Our data establish a novel experimental model for thymocyte-specific gene expression and suggest an important role for ZBP-89 in T cell development.

The upstream enhancer is necessary and sufficient for the expression of the pre-T cell receptor alpha gene in immature T lymphocytes.

The expression of the pre-T cell receptor alpha (pTa) gene occurs exclusively in immature T lymphocytes and is regulated by poorly defined mechanisms. We have analyzed the role of the upstream enhancer in pTa expression using conventional and bacterial artificial chromosome (BAC) reporter transgenes. The deletion of the enhancer completely abolished the expression of pTa BAC reporter in transgenic mice. Conversely, the combination of pTa enhancer and promoter targeted transgenes specifically to immature thymocytes, recapitulating the expression pattern of pTa. The core enhancer is conserved between mice and humans and contains a critical binding site for the transcription factor c-Myb. We also show that pTa promoter contains a conserved tandem E box site activated by E protein, HEB. These data establish the enhancer as a critical element regulating pTa gene expression and identify additional targets for c-Myb and E proteins in T cell development.

Structure and expression of the human immunoglobulin lambda genes.

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

An apoptosis-inducing isoform of neu differentiation factor (NDF) identified using a novel screen for dominant, apoptosis-inducing genes.

Apoptosis is a genetically programmed series of events that results in cell death. As a consequence, it is difficult to identify dominant genes that play a role in this process using genetic selections in conventional cell culture systems. Accordingly, we have established an efficient expression screen to isolate dominant, apoptosis-inducing genes. The assay is based on the apoptotic morphology induced in the human kidney cell line 293 after transient transfection of small plasmid pools from normalized cDNA expression libraries. Using this assay, we isolated a novel isoform of the proto-oncogene Neu differentiation factor (NDF), a ligand for erbB receptor tyrosine kinases. Several lines of experimental evidence indicate that this gene kills in a cell-autonomous fashion and independently of known erbB receptors. This apoptotic property of an NDF isoform is readily contrasted with NDF's transforming potential and might balance the tendency to tumorigenesis in cells that overexpress NDF.

The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation.

IP-10 is a member of the chemokine family of cytokines and is induced in a variety of cells in response to interferon gamma and lipopolysaccharide. The self-aggregation common to many chemokines, including IP-10, has hindered the identification of a specific IP-10 receptor. Using an IP-10 alkaline phosphatase fusion protein that fortuitously blocks this self-aggregation, we have identified an IP-10 binding site on a variety of cells including endothelial, epithelial, and hematopoietic cells. This binding site has a Kd of 25 nM, is inhibited by recombinant murine or human IP-10, and is dependent on the presence of cell surface heparan sulfate proteoglycans (HSPG). This conclusion is based on the findings that IP-10 binding to cells is: (a) inhibited by heparin and heparan sulfate; (b) sensitive to a 1 M NaCl wash; (c) eliminated by treatment with heparinase and trypsin; and (d) absent on mutant CHO cells that do not express cell surface HSPG. Platelet factor 4 (PF4), but not IL-8, monocyte chemoattractant protein-1, RANTES, monocyte inflammatory protein (MIP)-1 alpha, or MIP-1 beta, can compete effectively with IP-10 for binding to the cell surface. Furthermore, IP-10 shares with PF4 the ability to inhibit endothelial cell proliferation (IC50 = 150 nM). These studies demonstrate specificity in the interaction of chemokines and HSPG, and they define IP-10 and PF4 as a distinct subset of chemokines sharing an HSPG-binding site and angiostatic properties.

Molecular characterization of transgene-induced immunodeficiency in B-less mice using a novel quantitative limiting dilution polymerase chain reaction method.

B-less mice express a human immunoglobulin (Ig) lambda transgene that induces a severe deficiency of both immature pre-B and mature B lymphocytes. To understand this perturbation in B lymphopoiesis, we have devised a sensitive limiting dilution polymerase chain reaction assay that quantitates specific Ig rearrangements and thus quantitates B lineage cells at various stages of differentiation within unfractionated bone marrow. We find that there are significantly reduced frequencies of both VH-to-DJH and VK-to-JK rearrangements in the transgenic strain, whereas the frequency of D-to-JH rearrangements approximates that of wild type. Since Ig gene rearrangements occur in a stepwise fashion in which D-to-JH joining precedes that of VH-to-DJH and VK-to-JK, these results indicate that the major block of B lymphocyte development in the B-less strain occurs after D-to-JH rearrangement. Interestingly, sequence analysis of residual VHDJH junctions from transgenic pre-B lymphocytes reveals that an abnormally high proportion of these are out of frame and therefore nonproductive. Taken together, these data suggest that early expression of the transgenic lambda protein specifically prevents the development of a normal-sized population of precursor B lymphocytes coexpressing functional IgH. The transgene-induced immunodeficiency appears to arise by a precocious maturation process in which precursors bypass a developmental stage associated with cellular expansion.

Expression of c-MYC under the control of GATA-1 regulatory sequences causes erythroleukemia in transgenic mice.

To study oncogenesis in the erythroid lineage, we have generated transgenic mice carrying the human c-MYC proto-oncogene under the control of mouse GATA-1 regulatory sequences. Six transgenic lines expressed the transgene and displayed a clear oncogenic phenotype. Of these, five developed an early onset, rapidly progressive erythroleukemia that resulted in death of the founder animals 30-50 d after birth. Transgenic progeny of the sixth founder, while also expressing the transgene, remained asymptomatic for more than 8 mo, whereupon members of this line began to develop late onset erythroleukemia. The primary leukemic cells were transplantable into nude mice and syngeneic hosts. Cell lines were established from five of the six leukemic animals and these lines, designated erythroleukemia/c-MYC (EMY), displayed proerythroblast morphology and expressed markers characteristic of the erythroid lineage, including the erythropoietin receptor and beta-globin. Moreover, they also manifested a limited potential to differentiate in response to erythropoietin. Studies in the surviving transgenic line indicated that, contrary to our expectations, the transgene was not expressed in the mast cell lineage. That, coupled with the exclusive occurrence of erythroleukemia in all the transgenic lines, suggests that the GATA-1 promoter construct we have used includes regulatory sequences necessary for in vivo erythroid expression only. Additional sequences would appear to be required for expression in mast cells. Further, our results show that c-MYC can efficiently transform erythroid precursors if expressed at a vulnerable stage of their development.

Neu differentiation factor (NDF), a dominant oncogene, causes apoptosis in vitro and in vivo.

Neu differentiation factor (NDF, also called neuregulin) is a potent inducer of epithelial cell proliferation and has been shown to induce mammary carcinomas in transgenic mice. Notwithstanding this proliferative effect, we have shown that a novel isoform of NDF can induce apoptosis when overexpressed. Here we report that this property also extends to other NDF isoforms and that the cytoplasmic portion of NDF is largely responsible for the apoptotic effect, whereas the proliferative activity is likely to depend upon the secreted version of NDF. In accordance with these contradictory properties, we find that tumors induced by NDF display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.

Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung.

Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing.

B-less: a strain of profoundly B cell-deficient mice expressing a human lambda transgene.

We have created several transgenic mouse strains that bear the human lambda light chain gene driven by its own promoter and a mouse immunoglobulin heavy chain enhancer. The transgene is expressed in many tissues, with particularly high levels of expression in the bone marrow, thymus, spleen, and lymph nodes. One of these transgenic lines, B-less, displays a dramatic phenotype characterized by an acute susceptibility to bacterial and viral infections. Analysis of this strain shows it to be profoundly deficient in both immature (pre-B) and mature B cells, as well as in circulating immunoglobulin. The pre-B and B cell defects are cell autonomous, as judged by cell culture and bone marrow graft chimeras. Despite this B cell deficiency, the T cell lineage appears grossly normal as assessed by flow cytometric analysis and by its response to mitogen stimulation. Since an independently derived transgenic strain bearing the same human lambda construct displays a partial B-less phenotype, it is likely that the B lineage deficiency is due to a dominant effect of transgene expression rather than to the insertional perturbation of an endogenous mouse gene. It is interesting that the deficiency phenotype is fully expressed in the FVB/N genetic background, but is suppressed in F1 hybrids formed between the FVB/N and C57BL/6 inbred strains. Evidently, there are one or more dominant genetic suppressors of B-less in the C57BL/6 genome.

Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice.

To investigate the role of interleukin 7 (IL-7) in the development of the lymphoid system, we have generated two lines of transgenic mice carrying an IL-7 cDNA fused to an immunoglobulin heavy chain promoter and enhancer. This transgene is expressed in the bone marrow, lymph nodes, spleen, thymus, and skin provoking a perturbation of T cell development characterized by a marked reduction of CD4+ CD8+ (double-positive) thymocytes. Quite unexpectedly, however, both lines also develop a progressive cutaneous disorder involving a dermal lymphoid infiltrate that results in progressive alopecia, hyperkeratosis, and exfoliation. Although the infiltrate is primarily composed of T lineage cells, its development is not impeded in the athymic nu/nu background. Furthermore, the phenotype can be transmitted horizontally by transplanting lymphoid tissues or skin to syngeneic wild-type mice. Thus, the phenotype is conveyed by skin-homing, mobile cells (presumably the infiltrating lymphocytes) in a cell-autonomous fashion. In addition to the skin phenotype, this transgene also provokes the development of a lymphoproliferative disorder that induces B and T cell lymphomas within the first 4 mo of life. These findings suggest potential physiologic actions of IL-7 in T cell development and in cutaneous immunity. They also demonstrate that IL-7 can act as an oncogene in the living organism.

Allelic exclusion in transgenic mice carrying mutant human IgM genes.

Expression of the membrane-bound version of the human mu chain in transgenic mice results in the allelic exclusion of endogenous mouse Ig heavy chain genes (6). The secreted version of the human Ig transgene has no such effect. F1 hybrid animals that carry transgenes for both secreted and membrane-bound human mu chains produce both forms of the human heavy chain while strongly suppressing endogenous mouse mu expression. The simultaneous expression of the two rearranged transgenes in primary B cells suggests that allelic exclusion operates before the formation of a second functionally rearranged heavy chain gene in vivo.

Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia.

The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C-C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.

Nucleic acid and protein sequences of phosphocholine-binding light chains.

An 18-kilobase DNA fragment containing the sequence coding for both the variable and constant regions of the S107 mouse immunoglobulin light chain was cloned from total cellular DNA. The complete nucleotide sequence of the kappa-chain variable-region gene is reported. Determination of the amino acid sequence encoded by the DNA is found to be identical to the protein sequence of the T15 light chain through residue 88. Direct sequence analysis confirmed that the J1 joining segment is used in the recombination event producing the active kappa light chain gene.

Identification of three new Ig lambda-like genes in man.

Three new human lambda L chain-like Ig genes are identified by restriction enzyme and nucleotide sequence analysis. Two genes, 14.1 and 16.1, have intact J and C regions, and are potentially functional, with open reading frames. A third gene, 18.1, is a pseudogene. The evolutionary lineage of these genes compared to the known functional locus lambda C1-lambda C6 can be surmised from Southern blot and nucleotide homologies. This study demonstrates that the human lambda gene family is more complex than previously recognized.

Chromosomal location of human kappa and lambda immunoglobulin light chain constant region genes.

The chromosomal location of human constant region light chain immunoglobulin (Ig) genes has been determined by analyzing a group of human fibroblast/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human kappa and lambda constant region genes. Human chromosomes in each cell line were identified by isoenzyme analysis. The DNA from hybrid cells was digested with restriction endonucleases, size fractionated by gel electrophoresis, transferred to nitrocellulose or DBM paper, and hybridized with (32)P-labeled nucleic acid probes. The C(kappa) gene was assigned to human chromosome 2 and the C(lambda) genes to chromosome 22, based upon analysis of these hybrid cell lines, and these assignments were confirmed by analysis of subclones. A group of previously unassigned loci can be mapped to chromosome 2 by virtue of their close linkage to C(kappa). The lambda and kappa light chain and heavy chain Ig genes have now been assigned to all three human chromosomes that are involved in translocations with chromosome 8 in human B cell neoplasms. These techniques and probes provide a means to study the detailed arrangement of human Ig genes and their pseudogenes.