Carol C. Cheung

TAp73 knockout shows genomic instability with infertility and tumor suppressor functions

The Trp53 gene family member Trp73 encodes two major groups of protein isoforms, TAp73 and DeltaNp73, with opposing pro- and anti-apoptotic functions; consequently, their relative ratio regulates cell fate. However, the precise roles of p73 isoforms in cellular events such as tumor initiation, embryonic development, and cell death remain unclear. To determine which aspects of p73 function are attributable to the TAp73 isoforms, we generated and characterized mice in which exons encoding the TAp73 isoforms were specifically deleted to create a TAp73-deficient (TAp73(-/-)) mouse. Here we show that mice specifically lacking in TAp73 isoforms develop a phenotype intermediate between the phenotypes of Trp73(-/-) and Trp53(-/-) mice with respect to incidence of spontaneous and carcinogen-induced tumors, infertility, and aging, as well as hippocampal dysgenesis. In addition, cells from TAp73(-/-) mice exhibit genomic instability associated with enhanced aneuploidy, which may account for the increased incidence of spontaneous tumors observed in these mutants. Hence, TAp73 isoforms exert tumor-suppressive functions and indicate an emerging role for Trp73 in the maintenance of genomic stability.

Immunohistochemical Diagnosis of Papillary Thyroid Carcinoma

In thyroid, the diagnosis of papillary carcinoma (PC) is based on nuclear features; however, identification of these features is inconsistent and controversial. Proposed markers of PC include HBME-1, specific cytokeratins (CK) such as CK19, and ret, the latter reflecting a ret/PTC rearrangement. We applied immunohistochemical stains to determine the diagnostic accuracy of these three markers. Formalin-fixed, paraffin-embedded tissue from 232 surgically resected thyroid nodules included 40 hyperplastic nodules (NH), 35 follicular adenomas (FA), 138 papillary carcinomas (PC; 54 classical papillary tumors and 84 follicular variant papillary carcinomas [FVPC]), 4 follicular carcinomas (FC), 6 insular carcinomas (IC), 7 Hürthle cell carcinomas (HCC), and 2 anaplastic carcinomas (AC). HBME-1 and ret were negative in all NH and FA; some of these exhibited focal CK19 reactivity in areas of degeneration. Half of the FC and AC exhibited HBME-1 staining but no positivity for CK19 or ret. In PC, 20% of cases stained for all three markers. Classical PC had the highest positivity with staining for HBME-1 in 70%, CK19 in 80%, and ret in 78%. FVPC were positive for HBME-1 in 45%, for CK19 in 57%, and for ret in 63%; only 7 FVPC were negative for all three markers. The six IC exhibited 67% staining for HBME-1 and 50% positivity for CK19 and ret. The seven HCC had 29% positivity for HBME-1 and CK19, and 57% positivity for ret. This panel of three immunohistochemical markers provides a useful means of diagnosing PC. Focal CK19 staining may be found in benign lesions, but diffuse positivity is characteristic of PC. HBME-1 positivity indicates malignancy but not papillary differentiation. Only rarely are all three markers negative in PC; this panel therefore provides an objective and reproducible tool for the analysis of difficult thyroid nodules.

Interobserver Variability Between Expert Urologic Pathologists for Extraprostatic Extension and Surgical Margin Status in Radical Prostatectomy Specimens

Accurate Gleason score, pathologic stage, and surgical margin (SM) information is critical for the planning of post-radical prostatectomy management in patients with prostate cancer. Although interobserver variability for Gleason score among urologic pathologists has been well documented, such data for pathologic stage and SM assessment are limited. We report the first study to address interobserver variability in a group of expert pathologists concerning extraprostatic soft tissue (EPE) and SM interpretation for radical prostatectomy specimens. A panel of 3 urologic pathologists selected 6 groups of 10 slides designated as being positive, negative, or equivocal for either EPE or SM based on unanimous agreement. Twelve expert urologic pathologists, who were blinded to the panel diagnoses, reviewed 40× whole-slide scans and provided diagnoses for EPE and SM on each slide. On the basis of panel diagnoses, as the gold standard, specificity, sensitivity, and accuracy values were high for both EPE (87.5%, 95.0%, and 91.2%) and SM (97.5%, 83.3%, and 90.4%). Overall κ values for all 60 slides were 0.74 for SM and 0.63 for EPE. The κ values were higher for slides with definitive gold standard EPE (κ=0.81) and SM (κ=0.73) diagnoses when compared with the EPE (κ=0.29) and SM (κ=0.62) equivocal slides. This difference was markedly pronounced for EPE. Urologic pathologists show good to excellent agreement when evaluating EPE and SM. Interobserver variability for EPE and SM interpretation was principally related to the lack of a clearly definable prostatic capsule and crush/thermal artifact along the edge of the gland, respectively.

The Diagnostic Value of the Diatom Test for Drowning, I. Utility: A Retrospective Analysis of 771 Cases of Drowning in Ontario, Canada

The utility and validity of the diatom test for drowning was studied using a retrospective analysis of 771 cases of drowning mostly from Ontario, Canada, over the period 1977 to 1993. In this article (part one), the utility of the test was assessed using an analysis of test outcomes. In the companion article (part two), the validity of the test was assessed by analyzing the relationship between test outcome and characteristics of diatoms in the bone marrow and samples of putative drowning medium. In the present study, freshwater drownings accounted for 738 of the cases and 33 cases were drownings in bathtubs, pools, or toilets. Diatoms were recovered from the femoral bone marrow of 205 cases (28%) of freshwater drowning and four cases (12%) of domestic water drowning. There was a monthly variation in the frequency of positive test outcomes that could not be explained by seasonal differences in the total number of drownings. However, the monthly variation was strongly correlated with the periodic cycle of diatom blooms that occurs in freshwater. Positive diatom tests were characterized by a limited number of distinctive diatom species per case, and a restricted quantity and size range of diatom frustules. These results indicate that the diatom test for drowning will identify approximately one in three victims of freshwater drowning and may be useful in the assessment of deaths occurring in bathtubs. The correlation of the outcome of the diatom test for drowning with diatom blooms provides further evidence for the reliability of the test.

Disruption of the Lcn2 gene in mice suppresses primary mammary tumor formation but does not decrease lung metastasis

Based largely on studies in xenograft models, lipocalin-2 (Lcn2) has been implicated in the progression of multiple types of human tumors, including breast cancer. Here we examine the role of Lcn2 in mammary tumorigenesis and lung metastasis using an in vivo molecular genetics approach. We crossed a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with two independent gene-targeted Lcn2−/− mouse strains of the 129/Ola or C57BL/6 genetic background. The onset and progression of mammary tumor development and lung metastasis in the female progeny of these crosses were monitored over a 20-week period. Female Lcn2−/−MMTV-PyMT mice of the 129/Ola background (Lcn2−/−PyMT129) showed delayed onset of mammary tumors, and both Lcn2−/−PyMT129 mice and Lcn2−/−MMTV-PyMT mice of the C57BL/6 background (Lcn2−/−PyMTB6) exhibited significant decreases in multiplicity and tumor burden (∼2- to 3-fold), as measured by total tumor weight and volume. At the molecular level, mammary tumors derived from Lcn2−/−PyMTB6 females showed reduced matrix metalloproteinase-9 (MMP-9) activity and a lack of high molecular weight MMP activity. However, although increased MMP-9 activity has been linked to tumor progression, neither Lcn2−/−PyMTB6 nor Lcn2−/−PyMT129 female mice showed a reduction in lung metastases compared to Lcn2+/+PyMT controls. Our results demonstrate, using an in vivo animal model approach, that Lcn2 is a potent inducer of mammary tumor growth but not a significant promoter of lung metastasis.

Cross-talk between Chk1 and Chk2 in double-mutant thymocytes

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4−CD8− double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy.

Identification of BERP (brain-expressed RING finger protein) as a p53 target gene that modulates seizure susceptibility through interacting with GABAA receptors

p53 is a central player in responses to cellular stresses and a major tumor suppressor. The identification of unique molecules within the p53 signaling network can reveal functions of this important transcription factor. Here, we show that brain-expressed RING finger protein (BERP) is a gene whose expression is up-regulated in a p53-dependent manner in human cells and in mice. We generated BERP-deficient mice by gene targeting and demonstrated that they exhibit increased resistance to pentylenetetrazol-induced seizures. Electrophysiological and biochemical studies of cultured cortical neurons of BERP-deficient mice showed a decrease in the amplitude of GABAA receptor (GABAAR)-mediated miniature inhibitory postsynaptic currents as well as reduced surface protein expression of GABAARs containing the γ2-subunit. However, BERP deficiency did not decrease GABAARγ2 mRNA levels, raising the possibility that BERP may act at a posttranscriptional level to regulate the intracellular trafficking of GABAARs. Our results indicate that BERP is a unique p53-regulated gene and suggest a role for p53 within the central nervous system.

Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development

The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.