Wnt ligands influence tumour initiation by controlling the number of intestinal stem cells.

Many epithelial stem cell populations follow a pattern of stochastic stem cell divisions called 'neutral drift'. It is hypothesised that neutral competition between stem cells protects against the acquisition of deleterious mutations. Here we use a Porcupine inhibitor to reduce Wnt secretion at a dose where intestinal homoeostasis is maintained despite a reduction of Lgr5+ stem cells. Functionally, there is a marked acceleration in monoclonal conversion, so that crypts become rapidly derived from a single stem cell. Stem cells located further from the base are lost and the pool of competing stem cells is reduced. We tested whether this loss of stem cell competition would modify tumorigenesis. Reduction of Wnt ligand secretion accelerates fixation of Apc-deficient cells within the crypt leading to accelerated tumorigenesis. Therefore, ligand-based Wnt signalling influences the number of stem cells, fixation speed of Apc mutations and the speed and likelihood of adenoma formation.

CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer cell growth by activating Src family kinases.

The transmembrane glycoprotein, CUB (complement C1r/C1s, Uegf, Bmp1) domain-containing protein 1 (CDCP1) is overexpressed in several cancer types and is a predictor of poor prognosis for patients on standard of care therapies. Phosphorylation of CDCP1 tyrosine sites is induced upon loss of cell adhesion and is thought to be linked to metastatic potential of tumor cells. Using a tyrosine-phosphoproteomics screening approach, we characterized the phosphorylation state of CDCP1 across a panel of breast cancer cell lines. We focused on two phospho-tyrosine pTyr peptides of CDCP1, containing Tyr707 and Tyr806, which were identified in all six lines, with the human epidermal growth factor 2-positive HCC1954 cells showing a particularly high phosphorylation level. Pharmacological modulation of tyrosine phosphorylation indicated that, the Src family kinases (SFKs) were found to phosphorylate CDCP1 at Tyr707 and Tyr806 and play a critical role in CDCP1 activity. We demonstrated that CDCP1 overexpression in HEK293 cells increases global phosphotyrosine content, promotes anchorage-independent cell growth and activates several SFK members. Conversely, CDCP1 downregulation in multiple solid cancer cell lines decreased both cell growth and SFK activation. Analysis of primary human tumor samples demonstrated a correlation between CDCP1 expression, SFK and protein kinase C (PKC) activity. Taken together, our results suggest that CDCP1 overexpression could be an interesting therapeutic target in multiple solid cancers and a good biomarker to stratify patients who could benefit from an anti-SFK-targeted therapy. Our data also show that multiple tyrosine phosphorylation sites of CDCP1 are important for the functional regulation of SFKs in several tumor types.

Organizing posterior fossa hematomas simulating developmental cysts on prenatal imaging: report of 3 cases.

OBJECTIVE: We encountered 3 organizing tentorial hematomas simulating posterior fossa lesions such as Dandy-Walker, dermoid, or arachnoid cysts. We sought to correlate the clinical and pathologic features that allow distinction of developmental cysts from hematomas in the posterior fossa on imaging. METHODS. Prenatal sonograms in all fetuses and fetal magnetic resonance scans in 2 of the 3 were reviewed. One case proceeding to term had serial imaging up to age 11 months. Two cases had complete neuropathologic evaluation after termination. Maternal records were reviewed. RESULTS: In each case, the ultrasonographic findings were reminiscent of a developmental cyst but with echogenic debris, a rim, or both. Magnetic resonance imaging suggested tentorial hemorrhage in 2, 1 also with falcine hemorrhage. Serial prenatal and postnatal imaging showed resolution in the surviving case. Pathologically, 2 fetuses had organizing tentorial hematomas causing brain displacement. Calcifications, white matter damage, germinal matrix hemorrhage, and brain stem necrosis were also present. One mother had von Willebrand disease. CONCLUSIONS: Tentorial hematomas, with or without maternal coagulopathy, should be considered in the prenatal ultrasonographic diagnosis of cystlike posterior fossa abnormalities containing echogenic material. Fetal magnetic resonance imaging can suggest blood products. Hypoxic-ischemic brain damage may be concurrent; however, resolution of the hematoma, with no apparent neurologic sequelae, can occur.

Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects.

Astrocytomas are the leading cause of brain cancer in humans. Because these tumours are highly infiltrative, current treatments that rely on targeting the tumour mass are often ineffective. A mouse model for astrocytoma would be a powerful tool for dissecting tumour progression and testing therapeutics. Mouse models of astrocytoma have been designed to express oncogenic proteins in astrocytes, but have had limited success due to low tumour penetrance or limited tumour progression. We present here a mouse model of astrocytomas involving mutation of two tumour-suppressor genes, Nf1 and Trp53. Humans with mutations in NF1 develop neurofibromatosis type I (NF1) and have increased risk of optic gliomas, astrocytomas and glioblastomas. The TP53 tumour suppressor is often mutated in a subset of astrocytomas that develop at a young age and progress slowly to glioblastoma (termed secondary glioblastomas, in contrast to primary glioblastomas that develop rapidly de novo). This mouse model shows a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme, and may accurately model human secondary glioblastoma involving TP53 loss. This is the first reported mouse model of astrocytoma initiated by loss of tumour suppressors, rather than overexpression of transgenic oncogenes.

Mouse models of tumor development in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a prevalent familial cancer syndrome resulting from germ line mutations in the NF1 tumor suppressor gene. Hallmark features of the disease are the development of benign peripheral nerve sheath tumors (neurofibromas), which can progress to malignancy. Unlike humans, mice that are heterozygous for a mutation in Nf1 do not develop neurofibromas. However, as described here, chimeric mice composed in part of Nf1-/- cells do, which demonstrates that loss of the wild-type Nf1 allele is rate-limiting in tumor formation. In addition, mice that carry linked germ line mutations in Nf1 and p53 develop malignant peripheral nerve sheath tumors (MPNSTs), which supports a cooperative and causal role for p53 mutations in MPNST development. These two mouse models provide the means to address fundamental aspects of disease development and to test therapeutic strategies.

Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa.

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.

The intraoperative administration of ketorolac tromethamine in evaluating length of stay in a same day surgery unit.

This study was performed to determine if intraoperative administration of ketorolac tromethamine, a nonsteroidal anti-inflammatory drug, could shorten the length of stay in the same day surgery unit of a 535-bed medical center. Fifty laparoscopic patients were included in this study. Laparoscopic patients were chosen because they demonstrated an increased incidence of postoperative pain that delayed hospital discharge. The study population was divided into a test and control group. To eliminate procedural variability, the 50 subjects were chosen from a single group practice. Length of stay was recorded on a data retrieval form. Reasons for delay in discharge were identified. Subjects who received ketorolac tromethamine were discharged an average of 30 minutes earlier than those who did not receive it (94 minutes versus 124 minutes, respectively). A t-test revealed this difference to be statistically significant (P < or = .004). Intraoperative administration of ketorolac tromethamine should be considered for laparoscopy patients in same day surgery units, because criteria for discharge, such as stable vital signs, minimal nausea or vomiting, minimal pain, and oriented x 3 with a stable gait, are met sooner.

Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa.

We have found four mutations in the human gene encoding the beta-subunit of rod cGMP phosphodiesterase (PDE beta) that cosegregate with autosomal recessive retinitis pigmentosa, a degenerative disease of photoreceptors. In one family two affected siblings both carry allelic nonsense mutations at codons 298 and 531. Affected individuals have abnormal rod and cone electroretinograms. PDE beta is the second member of the phototransduction cascade besides rhodopsin that is absent or altered as a cause of retinitis pigmentosa, suggesting that other members of this pathway may be defective in other forms of this disease.

Proteolytic and metastatic activities of clones derived from a methylcholanthrene-induced murine fibrosarcoma.

Eighteen clones of a methylcholanthrene-induced murine fibrosarcoma (3AM) which were heterogeneous with respect to metastatic potentials and in vivo growth rates were examined for five different protease activities: acid protease (cathepsin D), BANA hydrolase (cathepsin B), neutral protease, collagenase, and plasminogen activator. Homogenates of the solid tumors produced by the clones were heterogeneous with respect to the activities of the proteases; these activities were in all cases (except plasminogen activator) higher than those obtained for normal muscle tissue. There was, however, no correlation between any of these protease activities and the metastatic potential or in vivo growth rates. The cathepsin B activity has also been evaluated on the cultured cells of the various clones. Results similar to that of the in vivo study were obtained. Analysis of the enzyme activity of the cell culture and of organ culture media, however, revealed no cathepsin B activity. It is concluded that the measurement of any one biochemical parameter such as proteolysis may not be sufficient to establish a correlation with the overall process of metastasis; a more precise dissection of the individual steps culminating in metastasis may provide a more fruitful approach to this problem.

Immune responsiveness against the human placenta. I. Generation of cellular and humoral activity in experimental animals.

Using a new adaptation of the 51Cr release assay, it was found that plasma membrane vesicles from the human placental microvillous surface were not only capable of eliciting both cellular and humoral immunity in mice, but were also susceptible to lysis by these immune components. The production of cytotoxic cells and circulating antibody in immunized animals was only observed in the presence of Freund's complete adjuvant. The cells exhibited reactivity only against vesicles from a limited range of placentae, while the antibodies showed reactions against vesicles from a wider range of placentae. These vesicles were also susceptible to lysis by antisera raised against placental alkaline phosphatase but were resistant to attack by antibodies raised against normal human serum and HLA (multispecific) determinants and by a cytotoxic monoclonal antibody against human beta 2-microglobulin. The cellular cytotoxicity in the spleens of immunized animals could be abrogated if the cells were pretreated with AKR anti-C3H antiserum and complement. Further, in the spleens of in immunized animals, a population of cells was detected that exhibited a 'natural' cytotoxicity against several of the membrane preparations. This cytotoxicity was heat-labile, being abrogated if the cells were preincubated at 37 degrees for 4 hr before the assay, but was resistant to attack by the anti-Thy 1 antiserum. The results indicate that the use of 51Cr-labelled placental microvillous vesicles is a useful way of searching for anti-trophoblast immunity which may now be applied to the search for immune responses in pregnancy.