Clinical Pharmacy Services in Canadian Emergency Departments: A 2022 National Survey.

Background: Support for the role of an emergency department (ED) clinical pharmacy team is evidence-based and recognized in numerous professional guidelines, yet previous literature suggests a low prevalence of ED clinical pharmacy services in Canadian hospitals. Objectives: To update (from a survey conducted in 2013) the description and quantification of clinical pharmacy services in Canadian EDs. Methods: All Canadian hospitals with an ED and at least 50 acute care beds were contacted to identify the presence of dedicated ED pharmacy services (defined as at least 0.5 full-time equivalent [FTE] position). Three separate electronic surveys were distributed by email to ED pharmacy team members (if available), pharmacy managers (at hospitals without an ED pharmacy team), and ED managers (all hospitals). The surveys were completed between November 2021 and January 2022. Results: Of the 254 hospitals identified, 117 (46%) had at least 0.5 FTE clinical pharmacy services in the ED (based on initial telephone screening). Of the 51 (44%) of 115 ED pharmacy team survey responses included in the analysis, 94% (48/51) had pharmacists and 55% (28/51) had pharmacy technicians. The majority of pharmacy managers and ED managers identified the need for ED pharmacy services where such services did not exist. Inadequate funding, competing priorities, and lack of training remain the most commonly reported barriers to providing this service. Personal safety concerns were reported by 20% (10/51) of respondents. Conclusions: Although the establishment of clinical pharmacy services in Canadian EDs has grown over the past 8 years, lack of funding and ED-specific training continue to limit this evidence-supported role in Canadian hospitals.

Contexte: La pratique de pharmacie clinique au service des urgences (SU) est fondée sur les données probantes et reconnue dans de nombreuses lignes directrices professionnelles. Cependant, des données portent à croire à une faible prévalence de ces services dans les SU des hôpitaux canadiens. Objectif: Mettre à jour la description et la quantification des services de pharmacie clinique dans les SU canadiens (à partir d'une enquête menée en 2013). Méthodes: Tous les hôpitaux canadiens dotés d'un SU et d'au moins 50 lits de soins de courte durée ont été contactés pour recenser la présence de services de pharmacie dédiés au SU (définis comme au moins 0,5 poste équivalent temps complet [ETC]). Trois sondages électroniques différents ont été distribués par courriel aux membres de l'équipe de la pharmacie du SU (le cas échéant), aux gestionnaires de pharmacie (dans les hôpitaux sans équipe de pharmacie au SU) et aux gestionnaires du SU (tous les hôpitaux). Les enquêtes ont été réalisées entre novembre 2021 et janvier 2022. Résultats: Sur les 254 hôpitaux recensés, 117 (46 %) disposaient d'au moins 0,5 ETC de services de pharmacie clinique au SU (d'après la sélection téléphonique initiale). Des 51 (44 %) sur 115 équipes de pharmacie du SU qui ont été inclus dans l'analyse, 94 % (48/51) avaient des pharmaciens et 55 % (28/51) avaient également du personnel technique en pharmacie. La majorité des directeurs de pharmacie et des directeurs des SU ont identifié le besoin de services de pharmacie au SU là où de tels services n'existent pas. Un financement inadéquat, des priorités concurrentes et le manque de formation demeurent les obstacles les plus souvent signalés à la prestation de ce service. Des problèmes de sécurité personnelle ont été mentionnés par 20 % (10/51) des répondants. Conclusion: Bien que l'établissement de services de pharmacie clinique dans les SU canadiens ait augmenté au cours des 8 dernières années, le manque de financement et de formation spécifique en pharmacie de médecine d'urgence continue de limiter ce rôle fondé sur des données probantes dans les hôpitaux canadiens.

Epithelial Ovarian Cancer: Providing Evidence of Predisposition Genes.

Epithelial ovarian cancer (EOC) is one of the cancers most influenced by hereditary factors. A fourth to a fifth of unselected EOC patients carry pathogenic variants (PVs) in a number of genes, the majority of which encode for proteins involved in DNA mismatch repair (MMR) pathways. PVs in BRCA1 and BRCA2 genes are responsible for a substantial fraction of hereditary EOC. In addition, PV genes involved in the MMR pathway account for 10-15% of hereditary EOC. The identification of women with homologous recombination (HR)-deficient EOCs has significant clinical implications, concerning chemotherapy regimen planning and development as well as the use of targeted therapies such as poly(ADP-ribose) polymerase (PARP) inhibitors. With several genes involved, the complexity of genetic testing increases. In this context, next-generation sequencing (NGS) allows testing for multiple genes simultaneously with a rapid turnaround time. In this review, we discuss the EOC risk assessment in the era of NGS.

Cell segmentation for immunofluorescence multiplexed images using two-stage domain adaptation and weakly labeled data for pre-training.

Cellular profiling with multiplexed immunofluorescence (MxIF) images can contribute to a more accurate patient stratification for immunotherapy. Accurate cell segmentation of the MxIF images is an essential step. We propose a deep learning pipeline to train a Mask R-CNN model (deep network) for cell segmentation using nuclear (DAPI) and membrane (Na+K+ATPase) stained images. We used two-stage domain adaptation by first using a weakly labeled dataset followed by fine-tuning with a manually annotated dataset. We validated our method against manual annotations on three different datasets. Our method yields comparable results to the multi-observer agreement on an ovarian cancer dataset and improves on state-of-the-art performance on a publicly available dataset of mouse pancreatic tissues. Our proposed method, using a weakly labeled dataset for pre-training, showed superior performance in all of our experiments. When using smaller training sample sizes for fine-tuning, the proposed method provided comparable performance to that obtained using much larger training sample sizes. Our results demonstrate that using two-stage domain adaptation with a weakly labeled dataset can effectively boost system performance, especially when using a small training sample size. We deployed the model as a plug-in to CellProfiler, a widely used software platform for cellular image analysis.

Non-Epithelial Ovarian Cancers: How Much Do We Really Know?

Non-epithelial ovarian cancers (NEOC) are a group of uncommon malignancies that mainly includes germ cell tumours (GCT), sex cord-stromal tumours (SCST), and some extremely rare tumours, such as small cell carcinomas and sarcomas. Each of these classifications encompasses multiple histologic subtypes. The aetiology and molecular origins of each sub-group of NEOC require further investigation, and our understanding on the genetic changes should be optimised. In this article, we provide an update on the clinical presentation, pathology, genetics, treatment and survival of the main histological subtypes of the GCT and the SCST, as well as of ovarian small cell carcinomas. We also discuss miRNA expression profiles of NEOC and report the currently active clinical trials that include NEOC.

Three-dimensional reconstruction of the orbital retrobulbar vasculature.

PURPOSE: The retrobulbar orbital vasculature is known to be complex and variable between individuals. This study aimed to produce a method to map the retrobulbar vessels, to generate 3D reconstructions, to visualise and to improve our understanding of their complexity. METHODS: Five human orbits donated under the Human Tissue Act (2004) were fixed in formalin, decalcified in 10% formic acid, and dehydrated in acetone at -20°C. Specimens were impregnated with epoxy resin, cured, and cut into 0.3 mm sections. Sections were stained with Gomori's trichrome stain, imaged, and reconstructed using 3D reconstruction software (BioVis3D, version 3.1). RESULTS: The arterial system was reconstructed in all five specimens. The superior ophthalmic vein (SOV) and the central retinal vein (CRV) were reconstructed in four specimens. E12 sheet plastination showed excellent results for histological analysis at a macroscopic level; however, anatomical topology was not entirely preserved on a microscopic level. Gomori's trichrome stain gave excellent results in highlighting axial sections of the arterial walls and their tunics, including finer calibre vessels, thus allowing detailed reconstruction of the arterial vasculature. Miller's stain for elastin showed poor results in differentiating vessels from soft tissue; venous vasculature was poorly identified with both stains. CONCLUSIONS: This study provided a detailed anatomical model of the retrobulbar orbital vascular system, a method that can be used for further studies to form a database relating to the topography of the arterial system. These models may be employed for teaching, and possible surgery planning, for both trainees and ophthalmic surgeons.

Quantitative single-cell analysis of immunofluorescence protein multiplex images illustrates biomarker spatial heterogeneity within breast cancer subtypes.

BACKGROUND: The extent of cellular heterogeneity in breast cancer could have potential impact on diagnosis and long-term outcome. However, pathology evaluation is limited to biomarker immunohistochemical staining and morphology of the bulk cancer. Inter-cellular heterogeneity of biomarkers is not usually assessed. As an initial evaluation of the extent of breast cancer cellular heterogeneity, we conducted quantitative and spatial imaging of Estrogen Receptor (ER), Progesterone Receptor (PR), Epidermal Growth Factor Receptor-2 (HER2), Ki67, TP53, CDKN1A (P21/WAF1), CDKN2A (P16INK4A), CD8 and CD20 of a tissue microarray (TMA) representing subtypes defined by St. Gallen surrogate classification. METHODS: Quantitative, single cell-based imaging was conducted using an Immunofluorescence protein multiplexing platform (MxIF) to study protein co-expression signatures and their spatial localization patterns. The range of MxIF intensity values of each protein marker was compared to the respective IHC score for the TMA core. Extent of heterogeneity in spatial neighborhoods was analyzed using co-occurrence matrix and Diversity Index measures. RESULTS: On the 101 cores from 59 cases studied, diverse expression levels and distributions were observed in MxIF measures of ER and PR among the hormonal receptor-positive tumor cores. As expected, Luminal A-like cancers exhibit higher proportions of cell groups that co-express ER and PR, while Luminal B-like (HER2-negative) cancers were composed of ER+, PR- groups. Proliferating cells defined by Ki67 positivity were mainly found in groups with PR-negative cells. Triple-Negative Breast Cancer (TNBC) exhibited the highest proliferative fraction and incidence of abnormal P53 and P16 expression. Among the tumors exhibiting P53 overexpression by immunohistochemistry, a group of TNBC was found with much higher MxIF-measured P53 signal intensity compared to HER2+, Luminal B-like and other TNBC cases. Densities of CD8 and CD20 cells were highest in HER2+ cancers. Spatial analysis demonstrated variability in heterogeneity in cellular neighborhoods in the cancer and the tumor microenvironment. CONCLUSIONS: Protein marker multiplexing and quantitative image analysis demonstrated marked heterogeneity in protein co-expression signatures and cellular arrangement within each breast cancer subtype. These refined descriptors of biomarker expressions and spatial patterns could be valuable in the development of more informative tools to guide diagnosis and treatment.

Heterogeneity of Circulating Tumor Cell-Associated Genomic Gains in Breast Cancer and Its Association with the Host Immune Response.

Tumor cells that preferentially enter circulation include the precursors of metastatic cancer. Previously, we characterized circulating tumor cells (CTC) from patients with breast cancer and identified a signature of genomic regions with recurrent copy-number gains. Through FISH, we now show that these CTC-associated regions are detected within the matched untreated primary tumors of these patients (21% to 69%, median 55.5%, n = 19). Furthermore, they are more prevalent in the metastases of patients who died from breast cancer after multiple rounds of treatment (70% to 100%, median 93%, samples n = 41). Diversity indices revealed that higher spatial heterogeneity for these regions within primary tumors is associated with increased dissemination and metastasis. An identified subclone with multiple regions gained (MRG clone) was enriched in a posttreatment primary breast carcinoma as well as multiple metastatic tumors and local breast recurrences obtained at autopsy, indicative of a distinct early subclone with the capability to resist multiple lines of treatment and eventually cause death. In addition, multiplex immunofluorescence revealed that tumor heterogeneity is significantly associated with the degree of infiltration of B lymphocytes in triple-negative breast cancer, a subtype with a large immune component. Collectively, these data reveal the functional potential of genetic subclones that comprise heterogeneous primary breast carcinomas and are selected for in CTCs and posttreatment breast cancer metastases. In addition, they uncover a relationship between tumor heterogeneity and host immune response in the tumor microenvironment. SIGNIFICANCE: As breast cancers progress, they become more heterogeneous for multiple regions amplified in circulating tumor cells, and intratumoral spatial heterogeneity is associated with the immune landscape.

School-based research agenda on healthcare simulation for nursing education in Hong Kong.

Background: Healthcare simulation has been used as a pedagogical strategy in nursing education. Evidence has shown one of the positive impacts that simulations replace clinical placement. These wide-ranging initiatives are essential, and they can guide a nursing school's simulation training. However, researching each innovation in the nursing field is beyond the scope. Methods: To focus our research and develop the capacity and capability to incorporate healthcare simulation in nursing education, we used a consensus building process to establish a school's research agenda. A modified Delphi process was adopted to reach a consensus among 10 nursing faculty members in one university with a visiting professor's support. Results: The three themes were identified as (1) embedding simulation into the baccalaureate in nursing curriculum, (2) designing effective simulation-based education and (3) simulating education in the broader world (adolescents). These themes were further categorised into two areas that used simulation in the educational and community settings. Sixty per cent of the faculty members agreed that the question, 'How can simulation be incorporated into clinical placements to enhance students' learning?' should be the highest research priority. Conclusion: This study adds understanding to incorporate simulation-based education in the nursing curriculum and community provides insights into future research.

Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.

Microultrasound molecular imaging of vascular endothelial growth factor receptor 2 in a mouse model of tumor angiogenesis.

High-frequency microultrasound imaging of tumor progression in mice enables noninvasive anatomic and functional imaging at excellent spatial and temporal resolution, although microultrasonography alone does not offer molecular scale data. In the current study, we investigated the use of microbubble ultrasound contrast agents bearing targeting ligands specific for molecular markers of tumor angiogenesis using high-frequency microultrasound imaging. A xenograft tumor model in the mouse was used to image vascular endothelial growth factor receptor 2 (VEGFR-2) expression with microbubbles conjugated to an anti-VEGFR-2 monoclonal antibody or an isotype control. Microultrasound imaging was accomplished at a center frequency of 40 MHz, which provided lateral and axial resolutions of 40 and 90 Im, respectively. The B-mode (two-dimensional mode) acoustic signal from microbubbles bound to the molecular target was determined by an ultrasound-based destruction-subtraction scheme. Quantification of the adherent microbubble fraction in nine tumor-bearing mice revealed significant retention of VEGFR-2-targeted microbubbles relative to control-targeted microbubbles. These data demonstrate that contrast-enhanced microultrasound imaging is a useful method for assessing molecular expression of tumor angiogenesis in mice at high resolution.

Detecting vascular changes in tumour xenografts using micro-ultrasound and micro-ct following treatment with VEGFR-2 blocking antibodies.

Blockade of vascular endothelial growth factor (VEGF) binding to its receptors on endothelial cells has been shown preclinically to induce tumour growth inhibition. Using ultrasound biomicroscopy (UBM) or micro-ultrasound imaging and micro-computed tomography (micro-CT) analysis, we have examined the effects of DC101, a highly specific vascular endothelial growth factor receptor-2 (VEGFR-2)-targeting antibody, in inducing growth inhibition and functional vascular changes in established melanoma (MeWo) xenografts in mice. Postprocessing of UBM imaging loops for speckle variance was introduced to estimate the level of functional blood flow in tumours. Perfused tumour area visualized by speckle variance revealed decreased blood flow within 48 h after DC101 injection (control versus DC101: 1.90 +/- 0.25% versus 1.01 +/- 0.11%, p < 0.01) and following a 3-wk DC101 therapy (control versus DC101: 0.76 +/- 0.14% versus 0.45 +/- 0.05%, p = 0.04), suggesting that VEGFR-2 blockade mediates both early and long-term effects on tumour blood flow. The growth of xenografts was significantly inhibited after treating with DC101 for 3 wk compared with controls. In addition to UBM, we examined the tumour vasculature in three-dimension (3D) using contrast-enhanced Micro-CT imaging, which displayed a reduction in the number of tumour vessels following extended VEGFR-2 blockade (vascular density of control versus DC101: 48.4 +/- 5.4% versus 20.6 +/- 1.8%). Lastly, decreased microvessel density (MVD) was noted in DC101-treated xenografts (3 wk) by performing immunohistochemical staining of endothelial marker CD34. Our study investigates tumour response to DC101 using complementing micro-ultrasound and micro-CT imaging tools.

Interframe clutter filtering for high frequency flow imaging.

This study investigates 'interframe' clutter filtering with a high frequency (HF) flow imaging system with the objective of improving the performance of HF microvascular imaging at high frame rates. An interframe filter exploits the correlation of tissue signals on the time scale of the frame rate and is, therefore, insensitive to tissue spectral broadening induced by sweeping a single element transducer over a region of tissue. In vitro experiments were conducted in a tissue-mimicking flow phantom over a range of mean flow velocities (0.5 to 70.0 mm/s). Power Doppler (PD) imaging and color flow (CF) imaging were performed for both slow (0.25 fps) and fast (20 fps) scanning acquisitions. Flow data acquired at 20 fps and interframe filtered had similar velocity and mean Doppler power values as the 0.25 fps single-frame filtered data sets. In vivo validation experiments were conducted using a 500 microm blood vessel in a human finger and detected blood flow of 2 to 3 mm/s. Further in vivo experiments examining experimental murine tumors demonstrated the feasibility of performing HF PD and CF imaging at high frame rates using interframe filtering.

PTEN in the haematopoietic system and its therapeutic indications.

A unique feature of the haematopoietic system is its self-renewal ability while maintaining a stable number of pluripotent haematopoietic stem cells (HSCs). Recently, two publications by Yilmaz and colleagues and Zhang and colleagues demonstrated that the loss of the tumour suppressor phosphatase and tensin homolog (PTEN) in mice disturbed the maintenance of quiescent HSCs and promoted leukemogenesis. Mammalian target of rapamycin (mTOR) inhibition with rapamycin distinctly rescued HSC development and depleted leukemic stem cells. Thus, the regulation of HSCs and leukemic cells seems to be governed by cell-context-dependent, PTEN-mediated regulation of mTOR.

Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors.

The contribution of bone marrow-derived circulating endothelial progenitor cells (CEPs) to tumor angiogenesis has been controversial, primarily because of their low numbers in blood vessels of untreated tumors. We show that treatment of tumor-bearing mice with vascular disrupting agents (VDAs) leads to an acute mobilization of CEPs, which home to the viable tumor rim that characteristically remains after such therapy. Disruption of this CEP spike by antiangiogenic drugs or by genetic manipulation resulted in marked reductions in tumor rim size and blood flow as well as enhanced VDA antitumor activity. These findings also provide a mechanistic rationale for the enhanced efficacy of VDAs when combined with antiangiogenic drugs.

Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia.

Because antiangiogenic therapies inhibit the growth of new tumor-associated blood vessels, as well as prune newly formed vasculature, they would be expected to reduce the supply of oxygen and thus increase tumor hypoxia. However, it is not clear if antiangiogenic treatments lead only to consistent and sustained increases in hypoxia, or transient decreases in tumor hypoxia along with periods of increased hypoxia. We undertook a detailed analysis of an orthotopically transplanted human breast carcinoma (MDA-MB-231) over a 3-week treatment period using DC101, an anti-vascular endothelial growth factor receptor 2 antibody. We observed consistent reductions in microvascular density, blood flow (measured by high-frequency micro-ultrasound), and perfusion. These effects resulted in an increase in the hypoxic tumor fraction, measured with an exogenous marker, pimonidazole, concurrent with an elevation in hypoxia-inducible factor-1alpha expression, an endogenous marker. The increase in tumor hypoxia was evident within 5 days and remained so throughout the entire course of treatment. Vascular perfusion and flow were impaired at days 2, 5, 7, 8, 14, and 21 after the first injection, but not at 4 hours. A modest increase in the vessel maturation index was detected after the 3-week treatment period, but this was not accompanied by an improvement in vascular function. These results suggest that sustained hypoxia and impairment of vascular function can be two consistent consequences of antiangiogenic drug treatment. The implications of the results are discussed, particularly with respect to how they relate to different theories for the counterintuitive chemosensitizing effects of antiangiogenic drugs, even when hypoxia is increased.

Three-dimensional ultrasound biomicroscopy for xenograft growth analysis.

We reported the use of high-frequency ultrasound biomicroscopy (UBM) in the quantitative analysis of early tumor growth in mice bearing melanoma xenografts in a noninvasive longitudinal assay. Initially, measurements of tumor width, depth and length were obtained using on-screen UBM calipers in real time and tumor volume was calculated with the standard ellipsoid formula w d l pi/6. We were able to detect initiating minute tumor nodules, with the lower limit of detection at approximately 0.01 mm(3) in volume. Successive parallel cross-sectional UBM images (33 microm step) encompassing the complete length of these tumors were also obtained and reconstructed into 3-D representations. Subsequent segmentational volumetric analysis provided a measure of tumor volume. Volume measurements using the two techniques were highly correlated when all 33 xenografts were studied (r = 0.9813, p < 0.0001) and a lower degree of correlation was measured with a subset of early small tumors (r = 0.7973, n = 16, p = 0.0004). Further analysis demonstrated that 3-D segmentational volumetric analysis yielded volume estimates that were often smaller than the caliper-and-formula calculation for most early developing xenografts. Thus, 3-D UBM imaging and segmentation is expected to be especially valuable for small tumors that were observed to grow in irregular shapes other than ellipsoids.

Brca2 deficiency does not impair mammary epithelium development but promotes mammary adenocarcinoma formation in p53(+/-) mutant mice.

Brca2 is an important tumor suppressor associated with susceptibility to breast cancer. Although increasing evidence indicates that the primary function of Brca2 is to facilitate the repair of DNA damage via the homologous recombination pathway, how Brca2 prevents breast cancer is largely unknown. To study the role of Brca2 specifically in mammary epithelium development, we crossed mice bearing the conditionally deficient allele Brca2(flox9-10) to mouse mammary tumor virus- or whey acidic protein-Cre transgenic lines. Analysis of these animals showed that Brca2 is not required for epithelial expansion in mammary glands of pregnant mice. In addition, examination of mammary gland involution revealed normal kinetics of mammary alveolar cell apoptosis after weaning of litters. Nevertheless, Brca2-deficient mice developed mammary adenocarcinomas after a long latency (average, 1.6 years). Detailed histopathological analysis of four of these tumors demonstrated that three of them showed abnormal p53 protein expression. A mutation in the p53 gene was detected in one case. Moreover, homozygosity versus heterozygosity for the Brca2 mutation heavily skewed the tumor spectrum toward mammary adenocarcinoma development in p53(+/-) mice. Our data indicate that Brca2 is not essential for mammary epithelium development but that Brca2 deficiency and down-regulated p53 expression can work jointly to promote mammary tumorigenesis.

Loss of Brca2 and p53 synergistically promotes genomic instability and deregulation of T-cell apoptosis.

BRCA2 is a breast cancer susceptibility gene of which the product is thought to be involved in monitoring genome integrity and cell cycle progression. Brca2-null mice have a defect in embryonic cellular proliferation and die in utero. Here we report the generation of T-cell lineage-specific Brca2-deficient (tBrca2(-/-)) mice using the Cre-loxP system. Mice with a flanked by loxP allele of Brca2 were crossed to transgenic mice bearing Cre recombinase driven by the T cell-specific promoter Lck. Thymic cellularity and distribution of subset populations were normal in tBrca2(-/-) mutants. Thymocytes from tBrca2(-/-) mice underwent normal apoptosis in response to a variety of stimuli, and activated tBrca2(-/-) T cells had normal proliferative capacity. tBrca2(-/-) T cells were more likely than wild-type cells to undergo spontaneous apoptosis, but apoptosed normally in response to restimulation or DNA-damaging stress signals. Examination of metaphase spreads of tBrca2(-/-) T cells revealed that the chromosomes often exhibited aberrations such as breaks and tri-radial structures. The level of chromosomal abnormalities was enhanced in T cells from tBrca2(-/-); p53(-/-) double-mutant mice. However, tBrca2(-/-); p53(-/-) T cells did not show the enhanced level of spontaneous apoptosis demonstrated by tBrca2(-/-) T cells, a difference that likely accounts for an increase in cell number and (3)[H]thymidine incorporation of double-mutant T cells in culture compared with either single mutant. Despite this increased T-cell number, the onset of T-cell lymphomas was only marginally accelerated in tBrca2(-/-); p53(-/-) mice compared with p53(-/-) mice. Our results support a role for Brca2 in repairing spontaneous DNA lesions, and suggest that loss of Brca2 enhances the susceptibility of mouse T-lineage cells to chromosomal aberrations and deregulation of apoptosis in the absence of p53.

Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner.

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.