Targeted disruption of Dkc1, the gene mutated in X-linked dyskeratosis congenita,causes embryonic lethality in mice

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome associated with increased cancer susceptibility. The X-linked form is due to mutations in the DKC1 gene encoding dyskerin, a nucleolar protein predicted to be involved in rRNA processing and associated with the telomerase complex. Available evidence suggests the pathology of DC is due to telomerase defects. We have used the inducible Cre/loxP system to produce deletions in the murine Dkc1 gene in early embryogenesis. A large deletion lacking exons 12-15 and a small deletion lacking only the last exon, were produced. We found both deletions showed a parent-of-origin effect with 100% embryonic lethality when the mutation occurred on the maternal Dkc1. Embryonic analysis at day E7.5 and E9.5 showed no male embryos carrying either deletion whereas females with maternally derived deletions died around day E9.5, with degeneration of the extra embryonic tissue, in which the paternal X-chromosome is inactivated. Female mice carrying the deletion in the paternally derived Dkc1 show extreme skewing of X-inactivation with the wild type X-chromosome active in all cells. Since mice with no telomerase are viable in the first generations the lethality we observe is unlikely to be due to the effects of mutated dyskerin on telomerase activity.     

Burkitt’s lymphoma variant of post-transplant lymphoproliferative disease (PTLD)

The occurrence of posttransplant lymphoproliferative disorder (PTLD) in solid organ allograft recipients can be quite varied in clinical presentation, histopathological characteristics and frequency. A variety of lymphomas can develop as a PTLD although some types appear infrequently and remain poorly understood in this clinical setting. In this report, we describe two cases of Burkitt s lymphoma presenting as a PTLD following liver transplantation. The recipients were 12 and 44 years of age and displayed gastrointestinal involvement by the tumors several years following transplant. The tumors displayed the typical histological features of Burkitt s lymphoma and were markedly positive for EBV. The tumors displayed similar immunophenotypic characteristics by flow cytometry and had rearrangements of the immunoglobulin J-H heavy chain. The tumors required aggressive chemotherapy and a cessation of immunosuppressive therapy. This report demonstrates that Burkitt s type lymphomas can develop in the posttransplant setting and that these tumors contain morphologic, cytofluorographic and molecular features identical to Burkitt s lymphomas that occur in non-transplant patients. Our experience is that these PTLD- Burkitt s lymphomas behave aggressively and require intensive chemotherapeutic intervention.     

Ductal lavage and ductoscopy: the opportunities and the limitations

Two related techniques of breast epithelial sampling have emerged in the past several years: ductal lavage, in which fluid-yielding nipple ducts are cannulated at their orifices and lavaged with saline while the breast is intermittently massaged; and ductoscopy, in which discharging or fluid-yielding duct orifices are dilated, intubated with a microendoscope, and the lumen directly visualized. Both of these techniques have significant potential in terms of allowing the repeated sampling of ductal epithelium over time and, as such, have generated considerable enthusiasm. However, data regarding the impact of these techniques on the detection of significant breast disease is very scant. It is important at the outset of the assessment of this new technology that breast cancer clinicians and clinical researchers think carefully about the standards of evidence that need to be met regarding the benefits of these procedures before they are widely adopted. In this review of the rationale and early results of these procedures, we attempt to define some of these evidentiary requirements.     

Che-1 affects cell growth by interfering with the recruitment of HDAC1 by Rb

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.     

Placenta growth factor-1 antagonizes VEGF-induced angiogenesis and tumor growth by the formation of functionally inactive PlGF-1/VEGF heterodimers

Tumor growth and metastasis require concomitant growth of new blood vessels, which are stimulated by angiogenic factors, including vascular endothelial growth factor (VEGF), secreted by most tumors. Whereas the angiogenic property and molecular mechanisms of VEGF have been well studied, the biological function of its related homolog, placenta growth factor (PlGF), is poorly understood. Here we demonstrate that PlGF-1, an alternatively spliced isoform of the PlGF gene, antagonizes VEGF-induced angiogenesis when both factors are coexpressed in murine fibrosarcoma cells. Overexpression of PlGF-1 in VEGF-producing tumor cells results in the formation of PlGF-1/VEGF heterodimers and depletion of the majority of mouse VEGF homodimers. The heterodimeric form of PlGF-1/VEGF lacks the ability to induce angiogenesis in vitro and in vivo. Similarly, PlGF-1/VEGF fails to activate the VEGFR-2-mediated signaling pathways. Further, PlGF-1 inhibits the growth of a murine fibrosarcoma by approximately 90% when PlGF-1-expressing tumor cells are implanted in syngeneic mice. In contrast, overexpression of human VEGF in murine tumor cells causes accelerated and exponential growth of primary fibrosarcomas and early hepatic metastases. Our data demonstrate that PlGF-1, a member of the VEGF family, acts as a natural antagonist of VEGF when both factors are synthesized in the same population of cells. The underlying mechanism is due to the formation of functionally inactive heterodimers.     

Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell lymphoma

The RUNX1/AML1 gene is a frequent target for chromosomal translocations in human leukemia. The biological properties of the resulting fusion products and the finding that haploinsufficiency increases the risk of developing leukemia (W-J. Song et al., Nat. Genet., 23: 166-175, 1999; M. Osata et al., Blood, 93: 1817-1824, 1999) have led to the widely held view that RUNX1 loss-of-function is a key event. However, we now report that the gene is a target for insertional mutagenesis in T-cell lymphomas of mice carrying a MYC oncogene, where promoter insertion results in overexpression without affecting the integrity of the coding sequence. Moreover, Runx1 haploinsufficiency does not accelerate lymphoma development in MYC/Runx2 transgenic or murine leukemia virus-infected mice. These findings reveal that the Runx1 gene can also act as a dominant oncogene and suggest that the involvement of the Runx gene family in human leukemia may be more widespread and complex than previously realized.     

Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods,and use in human biomonitoring

A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N(2)-yl)-7beta,8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, approximately 1.5 adducts/10(9) nucleotides using 20 micro g DNA) and a high signal-to-noise ratio (> or =100). The CIA BPDE-DNA standard curve gave 50% inhibition at 0.60 +/- 0.08 fmol BPdG (mean +/- SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-(3)H]BPDE was assayed by radiolabeling, (32)P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-(3)H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose-response was obtained with all assays. The BPDE-DNA CIA was further validated in MCL-5 cells exposed to 4 micro M BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE-DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH-DNA adducts/10(8) nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH-DNA adduct values obtained were highly correlated (r(2) = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.     

Seizures after spontaneous supratentorial intracerebral hemorrhage

PURPOSE: To characterize seizures after intracerebral hemorrhage (ICH), evaluating the risk of occurrence and relapse, predisposing factors, and prognostic significance, and to assess the utility of antiepileptic drug (AED) therapy as used in clinical practice. METHODS: The study sample consisted of 761 patients with spontaneous, nonaneurysmal, supratentorial ICH. Seizures were classified as immediate (within 24 h of ICH) and early (within 30 days of ICH). Baseline variables and clinical events were compared in the seizure and nonseizure group by using a multivariate regression model of failure time data. RESULTS: Fifty-seven patients had one or more seizures. The 30-day actuarial risk of a post-ICH seizure was 8.1%. Lobar location and small volume of ICH were independent predictors of immediate seizures. Early seizures were associated with lobar location and neurologic complications, mainly rebleeding. In patients with lobar ICH, the risk of early seizures was reduced by prophylactic AED therapy. Among seizure patients, history of alcohol abuse increased the risk of status epilepticus. Immediate and early seizures were not independent predictors of in-hospital mortality. CONCLUSIONS: Patients with ICH are exposed to a substantial risk of seizures; however, short-term mortality was not affected, and the risk of epilepsy was lower than previously thought. The likelihood of immediate seizures is influenced by factors that are inherent characteristics of ICH, whereas the chance of developing early seizures is influenced not only by certain characteristics of ICH, but also by unpredictable events. A brief period of therapy soon after ICH onset may reduce the risk of early seizures in patients with lobar hemorrhage.     

Comparison of the basal ganglia in rats, marmosets, macaques, baboons, and humans: volume and neuronal number for the output, internal relay, and striatal modulating nuclei

This study compares the basal ganglia of rats, marmosets, macaques, baboons, and humans. It uses established protocols to estimate the volume and number of neurons within the output nuclei (internal globus pallidus, IGP; and nondopaminergic substantia nigra, SNND), two internal relay and modulating nuclei (subthalamic nucleus, STh; and external globus pallidus, EGP), and a modulator of the striatum (dopaminergic substantia nigra, SND). Nuclear boundaries were defined by using immunohistochemistry for striatal afferents. Total numbers of Nissl-stained and parvalbumin-immunoreactive neurons were calculated by using the fractionator technique. Comparisons between species were standardized relative to brain mass (rats < marmosets < macaques < baboons < humans). The EGP consistently had more neurons relative to the IGP, STh, and SND, which had similar neuronal numbers within each species. The SNND had proportionally more neurons in rats than in primates (especially humans). The distribution of SND neurons varied substantially between rats and primates (very few ventrally located neurons in rats) with humans containing fewer SND neurons than other primates. The reduction in SND neurons in humans suggests less dopaminergic regulation of the basal ganglia system compared with other species. The consistency in the number of IGP neurons across all species, combined with the reduction in SNND neurons in humans, suggests a greater emphasis on output pathways through the IGP and that there are proportionally more STh and EGP neurons in humans.