Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.     

Carbonic anhydorase isoenzyme I (CA-I) concentration in feces and urine as a temporary marker of occult blood in beagle dogs.

This study was undertaken to investigate whether the concentration of carbonic anhydorase isoenzyme I (CA-I) in canine feces and urine is useful as a temporary marker of occult blood. Concentrations of CA-I were measured by enzyme-linked immunosorbent assay (ELISA). Fecal CA-I concentrations in 113 healthy beagle dogs (50 male and 63 female) of various ages ranged from 4.3 to 16.7 ng/g feces (mean; 7.0 +/- 2.9 ng/g feces). One milliliter of blood from 3 healthy beagle dogs was found to contain 1,047, 1,062 and 1,150 microg CA-I. The fecal CA-I concentrations of dogs receiving intragastric infusions of autologous blood (10 ml) were very low. However, the fecal CA-I concentrations of dogs receiving infusion of autologous blood (5 ml) into the ascending colon were very high. Detection of fecal CA-I would be useful for identifying dogs with hemorrhaging of the large intestine. Of 55 urinary samples collected from healthy beagle dogs by catheter, chemical tests for occult blood were negative in 44, but CA-I concentrations ranged from 1.8 to 12.6 ng/ml (mean; 6.9 +/- 5.4 ng/ml) by ELISA. The CA-I concentrations of the other 11 samples, which tested positive for occult blood on chemical testing, ranged from 41.2 to 525.0 ng/ml by ELISA. Although CA-I is not a specific marker of erythrocytes, CA-I may be used to detect occult blood in canine feces and urine until a specific immunological test kit using antibody for Hb is developed.     

The endosome-lysosome system in the absorptive cells of goldfish hindgut

Summary The vacuolar system in the absorptive cells of the goldfish hindgut was studied by rapid freeze-substituted and cytochemical techniques. The apical cytoplasm of the absorptive cells contained two types of vacuoles: endosomes and lysosomes. The former were characterized by an absence of acid phosphatase activity, a dot-like distribution of material at the peripheral rim, the labelling of the inner surface with horseradish peroxidase (HRP), and by frequent connections to cytoplasmic tubules (CT), which were also free of acid phosphatase activity. The latter vacuole was preferentially located in the deeper cytoplasm and was characterized by the presence of acid phosphatase activity, an electron-dense interior matrix, a peripheral electron-lucent region (a halo), and by the detachment of HRP from the inner surface. Connections between CTs and these latter vacuoles were rarely seen. In the deeper cytoplasm, fusion between endosomes and lysosomes was sometimes observed. These results suggest that the vacuoles which are associated with CTs are endosomes, but not lysosomes, and that internalized materials are transported through the endosome-lysosome system to a giant food vacuole in the cell.     

Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues

A critical and difficult part of studying cancer with DNA microarrays is data interpretation. Besides the need for data analysis algorithms, integration of additional information about genes might be useful. We performed genome-wide expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by reanalyzing a large collection of published DNA microarray datasets. We observed that the expression level of liver-specific genes in hepatocellular carcinoma (HCC) correlates with the clinically defined degree of tumor differentiation. Through unsupervised clustering of tissue-specific genes differentially expressed in tumors, we extracted expression patterns that are characteristic of individual cell types, uncovering differences in cell lineage among tumor subtypes. We were able to detect the expression signature of hepatocytes in HCC, neuron cells in medulloblastoma, glia cells in glioma, basal and luminal epithelial cells in breast tumors, and various cell types in lung cancer samples. We also demonstrated that tissue-specific expression signatures are useful in locating the origin of metastatic tumors. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage, and metastasis.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.