APOBEC signature mutation generates an oncogenic enhancer that drives LMO1 expression in T-ALL.

Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells, resulting in clonal selection. Although most cancer-causing mutations have been detected in the protein-coding regions of the cancer genome; driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well.

Identification of a cancer stem cell-specific function for the histone deacetylases, HDAC1 and HDAC7, in breast and ovarian cancer.

Tumours are comprised of a highly heterogeneous population of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumours in vivo. These cancer stem cells (CSCs) represent a significant clinical challenge as they are resistant to conventional cancer therapies and play essential roles in metastasis and tumour relapse. Despite this realization and great interest in CSCs, it has been difficult to develop CSC-targeted treatments due to our limited understanding of CSC biology. Here, we present evidence that specific histone deacetylases (HDACs) play essential roles in the CSC phenotype. Utilizing a novel CSC model, we discovered that the HDACs, HDAC1 and HDAC7, are specifically over-expressed in CSCs when compared to non-stem-tumour-cells (nsTCs). Furthermore, we determine that HDAC1 and HDAC7 are necessary to maintain CSCs, and that over-expression of HDAC7 is sufficient to augment the CSC phenotype. We also demonstrate that clinically available HDAC inhibitors (HDACi) targeting HDAC1 and HDAC7 can be used to preferentially target CSCs. These results provide actionable insights that can be rapidly translated into CSC-specific therapies.

The lived experience of visual creative expression for young adult cancer survivors.

Engaging in visual creative expression individually and in a therapeutic setting can be a beneficial experience for cancer survivors; however, most research in this field has been conducted with older adults. The current study aimed to address this gap by utilising van Manen's hermeneutic phenomenology to answer the following question: 'What is the lived experience and meaning of visual creative expression for young adult cancer survivors?' Seven young adults, diagnosed with cancer between the ages of 18 and 35, were interviewed about creative expression experiences, which they engaged in individually and/or in a therapeutic setting. Data analysis included a thematic reflection, guided existential reflection, and a process of writing and rewriting. Two superordinate themes were identified: increased self-understanding and a healing experience. Seven subthemes were also identified and included the following: being in the flow, allowing the body to express itself, renegotiating control, changing one's environment, being seen, respect for art as a separate entity and giving back. Findings suggest that visual creative expression can be a meaningful experience for young adult cancer survivors, and that this experience espouses both similarities and differences from experiences of older adult survivors. Recommendations are made for future research, in addition to implications for practitioners.

An unusual combination of diaphragmatic hernias in a patient presenting with the clinical features of restrictive pulmonary disease: report of a case.

The combination of a Morgagni hernia and a paraesophageal hernia in adults is very rarely encountered in clinical practice. In fact, to our knowledge, only three cases of this condition, which is probably a coincidental occurrence, have been reported in the medical literature. We discuss the management of a 74-year-old man found to have combined Morgagni and paraesophageal hernia who presented with clinical features of a restrictive pulmonary disease.

Genome-wide location and function of DNA binding proteins.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.

Insulin regulation of beta-cell function involves a feedback loop on SERCA gene expression, Ca(2+) homeostasis, and insulin expression and secretion.

The insulin receptor signaling pathway is present in beta-cells and is believed to be important in beta-cell function. We show here that insulin directly regulates beta-cell function in isolated rodent islets. Long-term insulin treatment caused a sustained increase in [Ca(2+)](i) and enhanced glucose-stimulated insulin secretion in rat islets, but failed to increase insulin content. Chronic activation of insulin receptor signaling by IRS-1 overexpression in the beta-cell inhibited gene expression of SERCA3, an endoplasmic reticulum Ca(2+)-ATPase. Insulin gene transcription was stimulated by insulin receptor signaling and insulin mimetic compound (L-783 281) in a glucose- and Grb2-dependent manner. Thus, beta-cell SERCA3 is a target for insulin regulation, which implies that beta-cell Ca(2+) homeostasis is regulated in an autocrine feedback loop by insulin. This study identifies a novel regulatory pathway of insulin secretion at the molecular level with two main components: (1) regulation of intracellular Ca(2+) homeostasis via SERCA3 and (2) regulation of insulin gene expression.

Osteopontin expression correlates with clinical outcome in patients with mycobacterial infection.

Osteopontin (OPN) is a protein that is expressed in chronic inflammatory diseases including tuberculosis, and its deficiency predisposes to more severe mycobacterial infections in mice. However, no reports have identified altered OPN expression in, or correlated these alterations to, infections in humans. The data presented herein identify alterations in the tissue expression of OPN protein and describe an inverse correlation between these levels and disease progression after inoculation of Mycobacterium bovis bacillus Calmette-Guérin vaccine in humans. Patients with regional adenitis and good clinical outcomes had abundant OPN in infected lymph nodes. This pattern of OPN accumulation was also observed in patients infected by M. avium-intracellulare. In contrast, patients with disseminated infection and histologically ill-defined granulomas had no significant osteopontin accumulation in infected lymph nodes; these patients had either deficiencies in the interferon-gamma receptor 1 or idiopathic immune defects. The level of OPN protein expression was inversely correlated with disseminated infection and, of particular interest, with death of the patient. We conclude that osteopontin expression correlates with an effective immune and inflammatory response when humans are challenged by a mycobacterial infection and that osteopontin contributes to human resistance against mycobacteria.

The yeast A kinases differentially regulate iron uptake and respiratory function.

Yeast has three A kinase catalytic subunits, which have greater than 75% identity and are encoded by the TPK genes (TPK1, TPK2, and TPK3) [Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. (1987) Cell 50, 277-287]. Although they are redundant for viability, the three A kinases are not redundant for pseudohyphal growth [Robertson, L. S. & Fink, G. R. (1998) Proc. Natl. Acad. Sci. USA 95, 13783-13787; Pan, X. & Heitman, J. (1999) Mol. Cell. Biol. 19, 4874-4887]; Tpk2, but not Tpk1 or Tpk3, is required for pseudohyphal growth. Genome-wide transcriptional profiling has revealed unique signatures for each of the three A kinases leading to the identification of additional functional diversity among these proteins. Tpk2 negatively regulates genes involved in iron uptake and positively regulates genes involved in trehalose degradation and water homeostasis. Tpk1 is required for the derepression of branched chain amino acid biosynthesis genes that seem to have a second role in the maintenance of iron levels and DNA stability within mitochondria. The fact that TPK2 mutants grow better than wild types on nonfermentable carbon sources and on media deficient in iron supports the unique role of Tpk2 in respiratory growth and carbon source use.

A frontal variant of Alzheimer's disease exhibits decreased calcium-independent phospholipase A2 activity in the prefrontal cortex.

A frontal variant of Alzheimer's disease (AD) has recently been identified on neuropathological and neuropsychological grounds (Johnson, J.K., Head, E., Kim, R., Starr, A., Cotman, C.W., 1999. Clinical and pathological evidence for a frontal variant of Alzheimer Disease. Arch. Neurol. 56, 1233-1239). Frontal AD differs strikingly from typical AD by the occurrence of neurofibrillary tangle densities in the frontal cortex as high or higher than in the entorhinal cortex. Since cerebrocortical membranes are commonly abnormal in Alzheimer's disease (AD), we assayed frontal AD cases for enzymes regulating membrane phospholipid composition. We specifically measured activity of phospholipase A2s (PLA2s) in dorsolateral prefrontal and lateral temporal cortices of frontal AD cases (n=12), which have respectively high and low densities of neurofibrillary tangles. In neither cortical area was Ca(2+)-dependent PLA2 activity abnormal compared to controls (n=12). In contrast, a significant 42% decrease in Ca(2+)-independent PLA2 activity was found in the dorsolateral prefrontal, but not the lateral temporal, cortex of the frontal AD cases. Similarly, the dorsolateral prefrontal cortex, but not the lateral temporal cortex of the frontal AD cases suffered a 42% decrease in total free fatty acid content, though neither that decrease nor those in any one species of free fatty acid was significant. The observed biochemical changes probably occurred in neurons given (a) our finding that PLA2 activity of cultured human NT2 neurons is virtually all Ca(2+)-independent and (b) the finding of others that nearly all Ca(2+)-independent PLA2 in brain gray matter is neuronal. The decrease in Ca(2+)-independent PLA2 activity is not readily attributable to Group VI or VIII iPLA2s since neither NT2N neurons nor our brain homogenates were greatly inhibited by drugs potently suppressing those iPLA2s. Decreased Ca(2+)-independent PLA2 activity in frontal AD may reflect a compensatory response to pathologically accelerated phospholipid metabolism early in the disorder. That could cause an early elevation of prefrontal free fatty acids, which can stimulate polymerization of tau and thus promote the prefrontal neurofibrillary tangle formation characteristic of frontal AD.

Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene.

Nucleic acid vaccines represent an attractive approach to generating antigen-specific immunity because of their stability and simplicity of delivery. However, there is still a need to increase the potency of DNA vaccines. Using human papillomavirus type 16 E7 as a model antigen, we evaluated the effect of linkage to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of antigen-specific immunity generated by naked DNA vaccines. We found that vaccines containing E7-HSP70 fusion genes increased the frequency of E7-specific CD8+ T cells by at least 30-fold relative to vaccines containing the wild-type E7 gene. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing tumors. Surprisingly, E7-HSP70 fusion vaccines exclusively targeted CD8+ T cells; immunological and antitumor effects were completely CD4-independent. These results indicate that fusion of HSP70 to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.

A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shock fusion proteins.

A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells.

Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin.

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused by Mycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected with Mycobacterium bovis BCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indicate that osteopontin augments the host response against a mycobacterial infection and that it acts independently from other antimycobacterial resistance mechanisms.

Increased antimycobacterial immunity in interleukin-10-deficient mice.

Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-gamma) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10(-/-)) mice should be more resistant to mycobacteria than control mice. Mycobacterium bovis bacillus Calmette-Guérin-infected IL-10(-/-) mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10(-/-) mice did not have increased levels of IFN-gamma, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10(-/-) mice produced increased levels of inflammatory cytokines, including IFN-gamma, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity.

Tumor necrosis factor alpha is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous system.

The pathogenesis of tuberculous meningitis, a devastating complication of tuberculosis in man, is poorly understood. We previously reported that rabbits with experimental tuberculous meningitis were protected from death by a combination of antibiotics and thalidomide therapy. Survival was associated with inhibition of tumor necrosis factor alpha (TNF-alpha) production by thalidomide. To test whether cerebrospinal fluid (CSF) levels of TNF-alpha correlated with pathogenesis, the response of rabbits infected in the central nervous system (CNS) with various mycobacterial strains was studied. CNS infection with Mycobacterium bovis Ravenel, M. bovis bacillus Calmette-Guérin (BCG) Pasteur, and M. bovis BCG Montreal were compared. M. bovis Ravenel induced the highest levels of TNF-alpha in the CSF in association with high leukocytosis, protein accumulation, and severe meningeal inflammation. BCG Pasteur had intermediate effects, and BCG Montreal was the least virulent. In addition, M. bovis Ravenel numbers were highest in the brain and CSF and the bacilli also disseminated more efficiently to distant organs, compared with BCG Pasteur and BCG Montreal. In subsequent experiments, rabbits were infected with either recombinant M. bovis BCG Montreal (vector), or BCG Montreal expressing the murine gene for TNF-alpha (BCG mTNF-alpha). BCG Montreal was rendered virulent by the expression of murine TNF-alpha, as demonstrated by high CSF leukocytosis, high protein accumulation, severe meningeal inflammation, persistent bacillary load, and progressive clinical deterioration. Taken together, these results demonstrate that the level of TNF-alpha produced during mycobacterial CNS infection determines, at least in part, the extent of pathogenesis.

Practice patterns of rural Texas physicians trained in a full-service family practice residency program.

The family practice residency program of John Peter Smith Hospital (JPSH) emphasizes "full-service" training to enable its graduates to provide a wide range of services to underserved areas, particularly rural communities. The purpose of this study was to investigate the practice patterns of rural JPSH family practice graduates. Of 180 identified JPSH family practice residency graduates who currently practice in rural Texas, 102 (57%) responded to a postcard survey. Data were analyzed by decade of graduation. Recent graduates were more likely to provide obstetrical care than were older graduates. The proportion of graduates providing obstetrical care at some time during their careers was similar. Recent graduates were more likely to perform office-based procedures such as colonoscopy and esophagogastroduodenoscopy. Rural JPSH family practice graduates provided more obstetrical care, critical care, and office-based procedures than did other rural members of the American Academy of Family Physicians. Practice patterns of JPSH family practice graduates have changed little over the past 30 years, except that recent graduates do more obstetrics and perform more endoscopies. These data show that the skills and knowledge of full-service family physicians are still needed in rural Texas.

An activator target in the RNA polymerase II holoenzyme.

Expression of protein-coding genes in eukaryotes involves the recruitment, by transcriptional activator proteins, of a transcription initiation apparatus consisting of greater than 50 polypeptides. Recent genetic and biochemical evidence in yeast suggests that a subset of these proteins, called SRB proteins, are likely targets for transcriptional activators. We demonstrate here, through affinity chromatography, photo-cross-linking, and surface plasmon resonance experiments, that the GAL4 activator interacts directly with the SRB4 subunit of the RNA polymerase II holoenzyme. The GAL4 activation domain binds to two essential segments of SRB4. The physiological relevance of this interaction is confirmed by mutations in SRB4, which occur within its GAL4-binding domain and which restore activation in vivo by a GAL4 derivative bearing a mutant activation domain.

Hematopoietic remodeling in interferon-gamma-deficient mice infected with mycobacteria.

Control of intracellular bacterial infections requires interferon-gamma (IFN-gamma) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN-gamma to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN-gamma-deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system. Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis. The splenic architecture of infected IFN-gamma-deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue. These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood. Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). These results suggest that in addition to its central role in cellular immunity, IFN-gamma may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis. This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway.

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.